Integrative Genome Viewer ( IGV ) snapshot showing ATAC - seq peaks along and upstream of the VWF gene in GPR56high vs . low samples ( top , average peak size of 10 GPR56high ( turquoise ) and 15 GPR56low samples ( violet ) ) , RNA - seq reads of the same location in AML samples with high ( n = 9 ) versus low ( n = 11 ) GPR56 expression ( two middle tracks ) , and RNA - seq reads of shLuc versus GPR56 knockdown CD34 + cells ( 3 bottom tracks , one of two replicates shown for each condition ) . Track height was group - scaled . Dashed vertical lines indicate binding sites for the annotated TFs . TFBS : transcription factor binding site derived from the HOCOMOCO v10 database ; TSS : transcription start site . TFs in blue bind to differentially accessible chromatin regions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Integrative Genome Viewer (IGV) snapshot showing ATAC-seq peaks along and upstream of the VWF gene in GPR56high vs. low samples (top, average peak size of 10 GPR56high (turquoise) and 15 GPR56low samples (violet)), RNA-seq reads of the same location in AML samples with high (n=9) versus low (n=11) GPR56 expression (two middle tracks), and RNA-seq reads of shLuc versus GPR56 knockdown CD34+ cells (3 bottom tracks, one of two replicates shown for each condition). Track height was group-scaled. Dashed vertical lines indicate binding sites for the annotated TFs. TFBS: transcription factor binding site derived from the HOCOMOCO v10 database; TSS: transcription start site. TFs in blue bind to differentially accessible chromatin regions.


Volcano plot of differential transcription factor ( TF ) motif accessibility ( activity ) in GPR56high ( turquoise ) vs GPR56low ( violet ) samples and their corresponding adjusted p - values determined with diffTF . Highlighted are TFs whose RNA expression was also positively or negatively affected by GPR56 KD in the RNA - seq dataset .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Volcano plot of differential transcription factor (TF) motif accessibility (activity) in GPR56high (turquoise) vs GPR56low (violet) samples and their corresponding adjusted p-values determined with diffTF. Highlighted are TFs whose RNA expression was also positively or negatively affected by GPR56 KD in the RNA-seq dataset.


Violin plots showing geometric mean ( horizontal line ) and individual values ( circles ) of the engraftment levels of different hematopoietic cell types in the bone marrow of NSG mice 20 weeks after transplantation of CB CD34 + cells following overnight - infection with shGPR56strong ( turqouise ) and shGPR56weak ( gray ) versus shLuc control ( violet ) . Shown are percentages of indicated populations , which are also co - positive for human CD45 and GFP , relative to all harvested bone marrow cells . HSPC : CD34 + SSClowCD33 - CD19 - , B precursor : CD34 + CD19 + CD33 - , myeloid progenitor ( myelo . prog . ) : CD34 + CD33 + CD19 - , mature B cells : CD19 + CD34 - , mature myeloid cells : CD33 + CD19 - CD34 - . Multiple unpaired t - tests of log - transformed values , p - values were Benjamini and Hochberg corrected ( padj ) , * padj < 0 . 05 , * * padj < 0 . 005 , * * * padj < 0 . 0005 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Violin plots showing geometric mean (horizontal line) and individual values (circles) of the engraftment levels of different hematopoietic cell types in the bone marrow of NSG mice 20 weeks after transplantation of CB CD34+ cells following overnight-infection with shGPR56strong (turqouise) and shGPR56weak (gray) versus shLuc control (violet). Shown are percentages of indicated populations, which are also co-positive for human CD45 and GFP, relative to all harvested bone marrow cells. HSPC: CD34+SSClowCD33-CD19-, B precursor: CD34+CD19+CD33-, myeloid progenitor (myelo. prog.): CD34+CD33+CD19-, mature B cells: CD19+CD34-, mature myeloid cells: CD33+CD19-CD34-. Multiple unpaired t-tests of log-transformed values, p-values were Benjamini and Hochberg corrected (padj), * padj<0.05, ** padj<0.005, *** padj<0.0005.


Adhesion of control and GPR56 KD K562 cells on fibronectin - functionalized substrates . Left : Overlay of phase contrast and microinterferometry images of control ( above ) and GPR56 KD cells ( below ) . The area of tight adhesion extracted by microinterferometry is highlighted in turquoise and violet , respectively . Right : Comparison of adhesion areas extracted by microinterferometry , reduction of adhesion area by factor 1 . 9 in GPR56 KD cells ( p = 1 . 0 x 10 - 10 , two - sided Mann - Whitney test , box plots showing medians , quartiles , and outliers according to the Tukey method ) . Technical replicates Ncontrol = 71 , Technical replicates NKD = 104 , scale bar 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Adhesion of control and GPR56 KD K562 cells on fibronectin-functionalized substrates. Left: Overlay of phase contrast and microinterferometry images of control (above) and GPR56 KD cells (below). The area of tight adhesion extracted by microinterferometry is highlighted in turquoise and violet, respectively. Right: Comparison of adhesion areas extracted by microinterferometry, reduction of adhesion area by factor 1.9 in GPR56 KD cells (p = 1.0 x 10-10, two-sided Mann-Whitney test, box plots showing medians, quartiles, and outliers according to the Tukey method). Technical replicates Ncontrol = 71, Technical replicates NKD = 104, scale bar 10 µm.


β - catenin protein expression in K562 cells infected with shGPR56strong or shLuc negative control in presence and absence of Wnt3a ( representative Western Blot ( above ) and quantification after normalization to GAPDH ( below ) . Shown is fold - change compared to shLuc in control media , bars and error bars represent mean and standard deviation of three biological replicates , unpaired t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

β-catenin protein expression in K562 cells infected with shGPR56strong or shLuc negative control in presence and absence of Wnt3a (representative Western Blot (above) and quantification after normalization to GAPDH (below). Shown is fold-change compared to shLuc in control media, bars and error bars represent mean and standard deviation of three biological replicates, unpaired t-test).


Left : Representative immunofluorescence ( IF ) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc ( above ) or shGPR56strong ( below ) . The non - motile primary cilium is visualized using antibodies against γ - tubulin , which stains the basal body , and the ciliary membrane marker ARL13B . DAPI indicates the nucleus . Images on the right show selected cells at 3 x magnification . Brightness was increased in all images to enhance visibility of cilia . Right : Percentage of RPE - 1 cells with primary cilium in shLuc vs . shGPR56strong RPE - 1 cells at different time points after the start of serum starvation . Unpaired t - test , bars and error bars represent mean and stdev of three biological replicate wells . * * * * p < 0 . 0001 , * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Left: Representative immunofluorescence (IF) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc (above) or shGPR56strong (below). The non-motile primary cilium is visualized using antibodies against γ-tubulin, which stains the basal body, and the ciliary membrane marker ARL13B. DAPI indicates the nucleus. Images on the right show selected cells at 3 x magnification. Brightness was increased in all images to enhance visibility of cilia. Right: Percentage of RPE-1 cells with primary cilium in shLuc vs. shGPR56strong RPE-1 cells at different time points after the start of serum starvation. Unpaired t-test, bars and error bars represent mean and stdev of three biological replicate wells. **** p<0.0001, *** p<0.0005, ** p<0.005, * p<0.05.


( A ) RT - qPCR analysis of NimB1 , NimB2 , NimB3 , NimB4 and NimB5 transcripts , normalized to RpL32 , from carcass , macrophages , fat body and gut of wild type ( w1118 ) L3 wandering larvae . Data are represented as mean ± SD from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) RT-qPCR analysis of NimB1, NimB2, NimB3, NimB4 and NimB5 transcripts, normalized to RpL32, from carcass, macrophages, fat body and gut of wild type (w1118) L3 wandering larvae. Data are represented as mean ± SD from three independent experiments.


( B ) Representative confocal imaging of NimB4 - sGFP macrophages of embryo at stage 16 ; lateral ( left ) and ventral ( right ) views ; Tissues were stained with anti - GFP ( green , corresponding to NimB4 - sGFP ) and anti - SIMU antibodies ( red , corresponding to macrophages ) . The arrows show the presence of NimB4 - sGFP inside the embryonic macrophages . Scale bars = 20

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative confocal imaging of NimB4-sGFP macrophages of embryo at stage 16; lateral (left) and ventral (right) views; Tissues were stained with anti-GFP (green, corresponding to NimB4-sGFP) and anti-SIMU antibodies (red, corresponding to macrophages). The arrows show the presence of NimB4-sGFP inside the embryonic macrophages. Scale bars = 20 μm


( A - C ) Representative confocal imaging of CFSE - stained apoptotic bodies ( green ) incubated with secreted NimB4 - RFP ( B , C ) or SPvkg - RFP ( A ) . Apoptotic bodies were pre - incubated in absence ( A , B ) or presence ( C ) of annexin V ( 25 μg / mL ) . Scale bar = 10 μm . ( D ) Quantification of the colocalization of NimB4 - RFP or SPvkg - RFP with the apoptotic bodies in presence or absence of Annexin V , as measured by Pearson ' s correlation coefficient . Values from at least four independent experiments are represented as mean ± SD ( * * P < 0 . 01 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Representative confocal imaging of CFSE-stained apoptotic bodies (green) incubated with secreted NimB4-RFP (B, C) or SPvkg-RFP (A). Apoptotic bodies were pre-incubated in absence (A, B) or presence (C) of annexin V (25 μg/mL). Scale bar = 10 μm. (D) Quantification of the colocalization of NimB4-RFP or SPvkg-RFP with the apoptotic bodies in presence or absence of Annexin V, as measured by Pearson's correlation coefficient. Values from at least four independent experiments are represented as mean ± SD (** P<0.01 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( A ) Representative confocal imaging of wild type ( w1118 ) , NimB4 sk2 and apoptosis - null NimB4 sk2 ; Df ( 3L ) H99 embryonic macrophages at stage 16 of development ; ventral and lateral view ; Tissues were co - stained with Dcp - 1 ( red , corresponding to apoptotic corpses ) and anti - SIMU ( green , corresponding to macrophages ) antibodies . The arrows show the presence of apoptotic cells inside embryonic macrophages . Scale bars = 20

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and apoptosis-null NimB4 sk2; Df(3L) H99 embryonic macrophages at stage 16 of development; ventral and lateral view; Tissues were co-stained with Dcp-1 (red, corresponding to apoptotic corpses) and anti-SIMU (green, corresponding to macrophages) antibodies. The arrows show the presence of apoptotic cells inside embryonic macrophages. Scale bars = 20 μm


( C ) Representative projections from confocal stacks of entire third instar wild type ( w1118 ) and NimB4 sk2 larval brains stained with anti - Draper ( green ) and anti - Dcp1 ( red ) . The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain . Scale bars = 100

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative projections from confocal stacks of entire third instar wild type (w1118) and NimB4 sk2 larval brains stained with anti-Draper (green) and anti-Dcp1 (red). The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain. Scale bars = 100 μm


( A ) Representative confocal imaging of wild type ( w1118 ) , NimB4 sk2 and draper macrophages incubated with fluorescently - labeled apoptotic cells ( red , CellTraceTM Red ) on ice for 1h and stained with AlexaFluorTM 488 phalloidin ( green ) . Scale bar : 10 μm . ( B ) Quantification of the number of apoptotic cells ( red ) binding to the macrophages ( green ) . Values from three independent experiments are represented as mean ± SD ( * * * P < 0 . 001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and draper macrophages incubated with fluorescently-labeled apoptotic cells (red, CellTraceTM Red) on ice for 1h and stained with AlexaFluorTM 488 phalloidin (green). Scale bar: 10 μm. (B) Quantification of the number of apoptotic cells (red) binding to the macrophages (green). Values from three independent experiments are represented as mean ± SD (*** P<0.001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( D ) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies . Wild type , NimB4sk2 , NimB4 genomic rescue ( NimB4sk2 , [ NimB4 ] ) , draper and NimC11 , eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ns : not significant ) . ( E ) Ex vivo phagocytosis assay using AlexFluor488 S . aureus Bioparticles . Wild type , NimB4sk2 and NimC11 , eater1 macrophages from L3 wandering were incubated with bioparticles for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies. Wild type, NimB4sk2, NimB4 genomic rescue (NimB4sk2, [NimB4]), draper and NimC11, eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05,** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests ns: not significant). (E) Ex vivo phagocytosis assay using AlexFluor488 S.aureus Bioparticles. Wild type, NimB4sk2 and NimC11, eater1 macrophages from L3 wandering were incubated with bioparticles for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (**** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( A ) Representative fluorescence microscopy images of wild type ( w1118 ) , NimB4sk2 , draper , eater1 and croquemort △ third instar larvae macrophages stained with LysoTracker Red ( live imaging ) . Overlay of fluorescence and differential interference contrast microscopy ( DIC ) . Scale bar = 10 μm . ( B ) Mean fluorescence intensity after staining with LysoTracker Red ( live confocal imaging ) . Values from at least three independent experiments are represented as mean ± SD ( * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative fluorescence microscopy images of wild type (w1118), NimB4sk2, draper, eater1 and croquemort△ third instar larvae macrophages stained with LysoTracker Red (live imaging). Overlay of fluorescence and differential interference contrast microscopy (DIC). Scale bar = 10 μm. (B) Mean fluorescence intensity after staining with LysoTracker Red (live confocal imaging). Values from at least three independent experiments are represented as mean ± SD ( ** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests).


( C ) Representative transmission electron micrographs of macrophages from wild type ( top , w1118 ) and NimB4sk2 ( bottom ) L3 wandering larvae . Scale bar : 2 μm ( D ) Quantification of the number of vesicles per macrophages in the electron micrographs . Values from at least three independent experiments are represented as mean ± SD ( * * * * P < 0 . 0001 by Mann - Whitney test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative transmission electron micrographs of macrophages from wild type (top, w1118) and NimB4sk2 (bottom) L3 wandering larvae. Scale bar: 2 μm (D) Quantification of the number of vesicles per macrophages in the electron micrographs. Values from at least three independent experiments are represented as mean ± SD (**** P<0.0001 by Mann-Whitney test).


( E ) Representative confocal imaging of Rab7EYFP immunostaining in wild type ( w1118 ) and NimB4 sk2 macrophages . Tissues were stained with anti - GFP ( red , Rab7 ) , counterstained with phalloidin ( gray ) and DAPI ( blue ) . The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages . Scale bar = 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Representative confocal imaging of Rab7EYFP immunostaining in wild type (w1118) and NimB4 sk2 macrophages. Tissues were stained with anti-GFP (red, Rab7), counterstained with phalloidin (gray) and DAPI (blue). The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages. Scale bar = 10 μm.


( A ) Representative confocal imaging of localization of Lamp1 - mcherry and LysoTracker Green in wild type ( w1118 ) , NimB4 sk2 and draper △ 5 macrophages ( live imaging ) . The arrows indicate the colocalization ( w1118 ) or the clustering ( NimB4sk2 and draper △ 5 ) of Lamp1 - mcherry and LysoTracker Green signals . Scale bar : 10 μm . ( B ) Quantification of the colocalization of Lamp1 - mcherry with LysoTracker Green , as measured by Pearson ' s correlation coefficient between the two signals . Values from at least five independent experiments are represented as mean ± SD ( * P < 0 . 05 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative confocal imaging of localization of Lamp1-mcherry and LysoTracker Green in wild type (w1118), NimB4 sk2 and draper△5 macrophages (live imaging). The arrows indicate the colocalization (w1118) or the clustering (NimB4sk2 and draper△5) of Lamp1-mcherry and LysoTracker Green signals. Scale bar: 10 μm. (B) Quantification of the colocalization of Lamp1-mcherry with LysoTracker Green, as measured by Pearson's correlation coefficient between the two signals. Values from at least five independent experiments are represented as mean ± SD (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests).


( C ) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo ™ Red . Wild type ( w1118 ) , NimB4sk2 and draper △ 5 macrophages from L3 wandering larvae were incubated with pHrodo ™ Red apoptotic cells for 60 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) . ( D ) Ex vivo phagocytosis assay using pHrodoTM Red S . aureus BioparticlesTM conjugates . Wild type , NimB4sk2 and NimC11 , eater1 Hml ∆ - Gal4 > UAS - GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S . aureus BioparticlesTM for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo™ Red. Wild type (w1118), NimB4sk2 and draper△5 macrophages from L3 wandering larvae were incubated with pHrodo™ Red apoptotic cells for 60 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). (D) Ex vivo phagocytosis assay using pHrodoTM Red S.aureus BioparticlesTM conjugates. Wild type, NimB4sk2 and NimC11, eater1 Hml∆-Gal4>UAS-GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S. aureus BioparticlesTM for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( A - B ) Representative fluorescence microscopy images of wild type ( A ) or NimB4sk2 ( B ) macrophages expressing Rab5 ( middle panel ) or Rab7 ( right panel ) driven by Hml ∆ - Gal4 and stained with the LysoTracker Red ( live imaging ) . Overlay of fluorescence and DIC . Scale bar = 10 μm . ( C ) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae ( confocal live imaging ) . Values from at least three independent experiments are represented as mean ± SD ( * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Representative fluorescence microscopy images of wild type (A) or NimB4sk2 (B) macrophages expressing Rab5 (middle panel) or Rab7 (right panel) driven by Hml∆-Gal4 and stained with the LysoTracker Red (live imaging). Overlay of fluorescence and DIC. Scale bar = 10 μm. (C) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae (confocal live imaging). Values from at least three independent experiments are represented as mean ± SD (** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


Same as ( E ) with amiRhen1 - 14 scions and non - transgenic WT or hen1 - 14 rootstocks . Average relative U6 - normalized band - intensity quantifications from two biological replicates are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Same as (E) with amiRhen1-14 scions and non-transgenic WT or hen1-14 rootstocks. Average relative U6-normalized band-intensity quantifications from two biological replicates are indicated.


Phenotype of pSUC2 : : amiRSUL plants in hst - 1 / - 25 / - 26 backgrounds .

Phenotype of pSUC2::amiRSUL plants in hst-1/-25/-26 backgrounds.


RT - qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes . Error - bars : SD . Welch ' s t - test p - values are indicated . n = 5 .

RT-qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes. Error-bars: SD. Welch's t-test p-values are indicated. n=5.


amiRSUL northern analysis in input and AGO1 - immunoprecipitated ( AGO1 - IP ) fractions isolated from leaves of pSUC2 : : amiRSUL plants in the specified genotypes . No Ab : No antibody added to the HST input extract ( IP negative control ) . miR165 / 166 and U6 were probed as endogenous controls . AGO1 western analysis and Coomassie blue ( Coom . ) staining of the western blot membrane are provided as controls . amiRSUL band - intensity quantifications , U6 - normalized for input samples , are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

amiRSUL northern analysis in input and AGO1-immunoprecipitated (AGO1-IP) fractions isolated from leaves of pSUC2::amiRSUL plants in the specified genotypes. No Ab: No antibody added to the HST input extract (IP negative control). miR165/166 and U6 were probed as endogenous controls. AGO1 western analysis and Coomassie blue (Coom.) staining of the western blot membrane are provided as controls. amiRSUL band-intensity quantifications, U6-normalized for input samples, are indicated.


amiRSUL northern analysis , in biological duplicates , in scions ( S ) and rootstocks ( R ) in the specified genotypes ' grafting combinations ( amiR : pSUC2 : : amiRSUL ) ( see also Appendix Fig . S12B ) . U6 : as in ( A ) . Average relative U6 - normalized band - intensity quantifications are indicated for each tissue .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

amiRSUL northern analysis, in biological duplicates, in scions (S) and rootstocks (R) in the specified genotypes' grafting combinations (amiR: pSUC2::amiRSUL) (see also Appendix Fig.S12B). U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated for each tissue.


amiRSUL northern analysis , in biological duplicates , from total RNA extracted from aphids fed on WT or amiR plants , in the specified genetic backgrounds . U6 : as in ( A ) . Average relative U6 - normalized band - intensity quantifications are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

amiRSUL northern analysis, in biological duplicates, from total RNA extracted from aphids fed on WT or amiR plants, in the specified genetic backgrounds. U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated.


Localization of HST : GFP and , as a reference , of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves ( C ) or roots ( D ) . CW : calcofluor white staining . Scale bars : 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Localization of HST:GFP and, as a reference, of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves (C) or roots (D). CW: calcofluor white staining. Scale bars: 20 µm.


Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2 : : HST : GFP , pSUC2 : : GFP or pSUC2 : : tmGFP9 . Scale bars : 100 µm . Confocal fluorescence images of the differentiation and division zones of pSUC2 : : GFPhst - 1 or pSUC2 : : tmGFP9hst - 1 root tips . Scale bars : 100 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2::HST:GFP, pSUC2::GFP or pSUC2::tmGFP9. Scale bars: 100 µm. Confocal fluorescence images of the differentiation and division zones of pSUC2::GFPhst-1 or pSUC2::tmGFP9hst-1 root tips. Scale bars: 100 µm.


Subcellular localizations of HST : GFP expressed from the UBQ10 promoter ( pUBQ ) in Arabidopsis roots ( i ) . Distinct planes of the same root cells imaged by confocal microscopy are shown ( ii - iii ) . PI : propidium iodide staining . Scale bars : 50µm ( i ) ; 10µm ( ii - iii ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Subcellular localizations of HST:GFP expressed from the UBQ10 promoter (pUBQ) in Arabidopsis roots (i). Distinct planes of the same root cells imaged by confocal microscopy are shown (ii-iii). PI: propidium iodide staining. Scale bars: 50µm (i); 10µm (ii-iii).


Subcellular localizations of hst - 3 : GFP expressed from pUBQ in root cells . Distinct planes of the same root cells imaged by confocal microscopy are shown ( i - ii ) . PI : as in ( A ) . Scale bars : 10µm .

Subcellular localizations of hst-3:GFP expressed from pUBQ in root cells. Distinct planes of the same root cells imaged by confocal microscopy are shown (i-ii). PI: as in (A). Scale bars: 10µm.


miR395 northern analysis in scions ( S ) and hyl1 - 2 rootstocks ( R ) , in the indicated genotypes ' combinations , under SO4 - sufficient ( + SO4 ) or SO4 - starved ( - SO4 ) conditions , in two biological replicates ( rep . ) . U6 was probed as an endogenous control . Relative U6 - normalized band - intensity quantifications are indicated for each tissue in - SO4 conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O

miR395 northern analysis in scions (S) and hyl1-2 rootstocks (R), in the indicated genotypes' combinations, under SO4-sufficient (+SO4) or SO4-starved (-SO4) conditions, in two biological replicates (rep.). U6 was probed as an endogenous control. Relative U6-normalized band-intensity quantifications are indicated for each tissue in -SO4 conditions.


miR160 northern analysis in Meselect - separated leaf vasculature and epidermis in WT versus hst - 1 plants , in three biological replicates ( rep . ) . U6 : as in ( A ) . Relative U6 - normalized band - intensity quantifications are indicated .

miR160 northern analysis in Meselect-separated leaf vasculature and epidermis in WT versus hst-1 plants, in three biological replicates (rep.). U6: as in (A). Relative U6-normalized band-intensity quantifications are indicated.


Basic fuchsin staining of protoxylem ( unfilled arrowheads ) and metaxylem ( filled arrowheads ) in WT , shr - 2 or hst - 1 roots . * : protoxylem gap . Scale bars : 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Basic fuchsin staining of protoxylem (unfilled arrowheads) and metaxylem (filled arrowheads) in WT, shr-2 or hst-1 roots. *: protoxylem gap. Scale bars: 10 µm.


B . Survival frequencies observed after DSB induction at TEL6R in WT , dnl4Δ , sir4Δ cells , expressing or not high levels of Sir3p ( oeSir3 and WT respectively ) . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . C . Survival frequencies observed after DSB induction at LYS2 in the indicated strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Survival frequencies observed after DSB induction at TEL6R in WT, dnl4Δ, sir4Δ cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Error bars indicate survival standard error (SEM) of at least three independent experiments. C. Survival frequencies observed after DSB induction at LYS2 in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


F . Representative images of Mre11 - YFP foci in response to an I - SceI - induced DSB at LYS2 in WT cells , expressing or not high levels of Sir3p ( oeSir3 and WT respectively ) . Scale bars are 2 μm . G . Quantification of cells with DSB induced Mre11 - YFP foci after DSB induction at LYS2 I - SceI cleavage site in WT , sae2Δ and Sir3 overexpressing ( oeSir3 ) strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Representative images of Mre11-YFP foci in response to an I-SceI-induced DSB at LYS2 in WT cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Scale bars are 2 μm. G. Quantification of cells with DSB induced Mre11-YFP foci after DSB induction at LYS2 I-SceI cleavage site in WT, sae2Δ and Sir3 overexpressing (oeSir3) strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


H . Survival frequencies after DSB induction at LYS2 locus , in strains where SIR3 is expressed from its native , pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid . Fold increase in Sir3 protein by pAHD1 or pGPD is indicated . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Survival frequencies after DSB induction at LYS2 locus, in strains where SIR3 is expressed from its native, pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid. Fold increase in Sir3 protein by pAHD1 or pGPD is indicated. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


A . Representative images of Sir3 - mCherry and Sae2 - GFP signal in WT and SIR3 overexpressing cells . Scale bars are 2 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative images of Sir3-mCherry and Sae2-GFP signal in WT and SIR3 overexpressing cells. Scale bars are 2 μm.


B . Sir3 - binding at TEL6R in untagged , WT , sir3Δ cells or in cells overexpressing Sir3 ( oeSir3 ) . Binding is probed by ChIP - qPCR 0 . 2 ( red arrows ) and 1kb ( blue arrows ) from telomeres and at the OGG1 control locus using antibodies against Sae2 - GFP . The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates ( SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Sir3-binding at TEL6R in untagged, WT, sir3Δ cells or in cells overexpressing Sir3 (oeSir3). Binding is probed by ChIP-qPCR 0.2 (red arrows) and 1kb (blue arrows) from telomeres and at the OGG1 control locus using antibodies against Sae2-GFP. The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates (SEM).


C . Co - immunoprecipitation between Sir3 and Sae2 - GFP from cells overexpressing Sir3 , analysed by Western blot with anti - GFP and anti - Sir3 antibodies . D . Co - immunoprecipitation between Sir3 and Sae2 - GFP from WT cells using antibodies against Sae2 - GFP , analysed by Western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Co-immunoprecipitation between Sir3 and Sae2-GFP from cells overexpressing Sir3, analysed by Western blot with anti-GFP and anti-Sir3 antibodies. D. Co-immunoprecipitation between Sir3 and Sae2-GFP from WT cells using antibodies against Sae2-GFP, analysed by Western blot.


D . Yeast two - hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells . Growth on - His + 3AT and blue coloration on X - gal indicate an interaction .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O

D. Yeast two-hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells. Growth on -His + 3AT and blue coloration on X-gal indicate an interaction.


B . Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C . C . Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C .

B. Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C. C. Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C.


A . Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


B . Representative images of Sae2 - GFP in WT cells and in cells overexpressing either full - length Sir3 or the sir3SaID domain . Scale bars are 2 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Representative images of Sae2-GFP in WT cells and in cells overexpressing either full-length Sir3 or the sir3SaID domain. Scale bars are 2 μm.


F . Representative silver - stained gels of in vitro GST - pulldown of GST or GST - Sir3SaID , GST - sir3 - T557ISaID and Sae2C purified peptides . Control : Sae2C ( 300 ng , lane 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Representative silver-stained gels of in vitro GST-pulldown of GST or GST-Sir3SaID, GST-sir3-T557ISaID and Sae2C purified peptides. Control: Sae2C (300 ng, lane 6).


G . Co - immunoprecipitation between Sae2 - GFP and Sir3 from untagged , Sae2 - GFP WT cells , and Sae2 - GFP cells overexpressing WT Sir3 ( oeSir3 , WT ) , Sae2 - GFP sir3Δ or Sae2 - GFP overexpressing the sir3 - T557I mutant ( oeSir3 , T557I ) using antibodies against Sae2 - GFP , analysed by Western blot with anti - GFP and anti - Sir3 antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Co-immunoprecipitation between Sae2-GFP and Sir3 from untagged, Sae2-GFP WT cells, and Sae2-GFP cells overexpressing WT Sir3 (oeSir3, WT), Sae2-GFP sir3Δ or Sae2-GFP overexpressing the sir3-T557I mutant (oeSir3, T557I) using antibodies against Sae2-GFP, analysed by Western blot with anti-GFP and anti-Sir3 antibodies.


A . Survival frequencies after DSB induction at LYS2 locus in the indicated strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

A. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


C . Survival frequencies after DSB induction at LYS2 locus in the indicated strains . Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression . Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression. Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


E . Representative images of two hybrid assays testing the interaction between the full - length Sir3 and full - length Sae2 proteins in WT cells expressing or not high levels of Sir4 ( oeSir4 and WT respectively ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative images of two hybrid assays testing the interaction between the full-length Sir3 and full-length Sae2 proteins in WT cells expressing or not high levels of Sir4 (oeSir4 and WT respectively).


A . Relative fold enrichment of Sae2 at 0 . 2 kb from I - SceI site was evaluated by qPCR after ChIP with anti - GFP antibodies . The error bars indicate the variation between at least three biological replicas ( SEM ) . B . Relative fold enrichment of Sae2 at 0 . 2 kb from TEL6R after DSB induction at the LYS2 I - SceI site was evaluated by qPCR after ChIP with anti - GFP antibodies . The error bars indicate the variation between at least three biological replicas ( SEM ) . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Relative fold enrichment of Sae2 at 0.2 kb from I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). B. Relative fold enrichment of Sae2 at 0.2 kb from TEL6R after DSB induction at the LYS2 I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


D . Survival frequencies after DSB induction at LYS2 locus in sae2 - E267E mutant cells overexpressing or not full - length Sir3 ( oeSir3 ) . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Survival frequencies after DSB induction at LYS2 locus in sae2-E267E mutant cells overexpressing or not full-length Sir3 (oeSir3). Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


Schematic , western blot , and immunofluorescence analysis of the 3 × FLAG - UBXD8 knockin U2OS cell line . Scale bar , 5 μm . Data information : In C , mitochondria were visualized by anti - Tom20 immunostaining ; ER was labeled by anti - PDI immunostaining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Schematic, western blot, and immunofluorescence analysis of the 3×FLAG-UBXD8 knockin U2OS cell line. Scale bar, 5 μm. Data information: In C, mitochondria were visualized by anti-Tom20 immunostaining; ER was labeled by anti-PDI immunostaining.


Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆ UBXD8 HeLa cells .

Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆UBXD8 HeLa cells.


Immunoprecipitation analysis of UBXD8 interaction with mitochondrial ( MARCH5 and MUL1 ) and ER ( RNF185 ) ubiquitin E3 ligases . HeLa cells were infected with lentivirus to stably express HA - tagged ubiquitin E3 ligases . Whole cell lysates were prepared for anti - HA immunoprecipitation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoprecipitation analysis of UBXD8 interaction with mitochondrial (MARCH5 and MUL1) and ER (RNF185) ubiquitin E3 ligases. HeLa cells were infected with lentivirus to stably express HA-tagged ubiquitin E3 ligases. Whole cell lysates were prepared for anti-HA immunoprecipitation.


Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185 . Whole cell lysates from 3 × FLAG - UBXD8 knockin HEK293T cells were prepared for anti - FLAG immunoprecipitation . MARCH5 and RNF185 : short exposure for whole cell lysate blots , long exposure for FLAG - IP blots .

Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185. Whole cell lysates from 3×FLAG-UBXD8 knockin HEK293T cells were prepared for anti-FLAG immunoprecipitation. MARCH5 and RNF185: short exposure for whole cell lysate blots, long exposure for FLAG-IP blots.


Fractionation analysis of UBXD8 ' s effect on VCP recruitment to mitochondria and the ER . Whole cell lysate , mitochondria , and ER fractions were purified from WT , ∆ UBXD8 , and ∆ UBXD8 HeLa cells rescued with UBXD8 or the UBX * mutant . UBX * - UBXD8 carries four point mutations ( K367A , F407A , P408G , R409A ) in the UBX domain to disrupt the UBXD8 - VCP interaction . For each sample , 5 μg proteins were loaded for western blot analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fractionation analysis of UBXD8's effect on VCP recruitment to mitochondria and the ER. Whole cell lysate, mitochondria, and ER fractions were purified from WT, ∆UBXD8, and ∆UBXD8 HeLa cells rescued with UBXD8 or the UBX* mutant. UBX*-UBXD8 carries four point mutations (K367A, F407A, P408G, R409A) in the UBX domain to disrupt the UBXD8-VCP interaction. For each sample, 5 μg proteins were loaded for western blot analysis.


Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells . Data are shown as mean ± SE from three biological repeats . Statistics : one - way ANOVA ; * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells. Data are shown as mean ± SE from three biological repeats. Statistics: one-way ANOVA; **P < 0.01, ***P < 0.001.


A , B . Western blot ( A ) and quantitative ( B ) analyses of MiD49 and Mcl1 degradation in WT and ∆ MARCH5 HeLa cells . Cycloheximide ( CHX : 200 μg / ml ; protein synthesis inhibitor ) ; MG132 : 20 μM , proteasome inhibitor . Data information : Data are shown as mean ± SE from three biological repeats ( B , Statistics : two - tailed unpaired Student ' s t - test ( B , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. Western blot (A) and quantitative (B) analyses of MiD49 and Mcl1 degradation in WT and ∆MARCH5 HeLa cells. Cycloheximide (CHX: 200 μg/ml; protein synthesis inhibitor); MG132: 20 μM, proteasome inhibitor. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


C , D . Western blot ( C ) and quantitative ( D ) analyses of Mcl1 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats D , Statistics : two - tailed unpaired Student ' s t - test D , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. Western blot (C) and quantitative (D) analyses of Mcl1 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


E , F . Western blot ( E ) and quantitative ( F ) analyses of MiD49 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats F ) . Statistics : two - tailed unpaired Student ' s t - test F ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F. Western blot (E) and quantitative (F) analyses of MiD49 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats F). Statistics: two-tailed unpaired Student's t-test F); *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


Fractionation analysis of the subcellular localization of mito - UBXD8 and ER - UBXD8 . Whole cell lysate , mitochondria , and ER fractions were purified from the indicated HeLa cells . Exogenously - expressed mito - UBXD8 has similar expression level with endogenous UBXD8 in WT cells ; exogenously - expressed WT UBXD8 and ER - UBXD8 have similar expression levels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fractionation analysis of the subcellular localization of mito-UBXD8 and ER-UBXD8. Whole cell lysate, mitochondria, and ER fractions were purified from the indicated HeLa cells. Exogenously-expressed mito-UBXD8 has similar expression level with endogenous UBXD8 in WT cells; exogenously-expressed WT UBXD8 and ER-UBXD8 have similar expression levels.


D , E . Western blot ( D ) and quantitative ( E ) analyses of Mcl1 degradation in the indicated HeLa cells . ∆ UBXD8 HeLa cells were rescued with vector , WT - , mito - , and ER - UBXD8 . Data are shown as mean ± SE from three biological repeats ( E ) . Statistics : two - tailed unpaired Student ' s t - test ( E ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E. Western blot (D) and quantitative (E) analyses of Mcl1 degradation in the indicated HeLa cells. ∆UBXD8 HeLa cells were rescued with vector, WT-, mito-, and ER-UBXD8. Data are shown as mean ± SE from three biological repeats (E). Statistics: two-tailed unpaired Student's t-test (E); *P < 0.05, **P < 0.01, ***P < 0.001.


Cell survival analysis of the indicated HeLa cells treated with Actinomycin D ( ActD , 1 μM ) or Doxorubicin ( Doxo , 10 μM ) . Representative images are shown . Cell viability was measured by Cell Titer - Glo . Scale bar , 50 μm . Data information : Data are shown as mean ± SE from three biological repeats D , Statistics : two - tailed unpaired Student ' s t - test D , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell survival analysis of the indicated HeLa cells treated with Actinomycin D (ActD, 1 μM) or Doxorubicin (Doxo, 10 μM). Representative images are shown. Cell viability was measured by Cell Titer-Glo. Scale bar, 50 μm. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001.


Western blot analysis of apoptosis activation ( caspase cleavage and PARP cleavage ) in the indicated HeLa cells treated similarly as in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of apoptosis activation (caspase cleavage and PARP cleavage) in the indicated HeLa cells treated similarly as in (D).


Cell survival analysis of the indicated HeLa cells . 7 × 105 cells in one well of 6 - well plate were treated with ActD ( 200 nM ) for 4 hours or Doxo ( 50 nM ) for 2 hours and subsequently cultured in normal medium for 21 days . Surviving colonies were stained by 0 . 1 % ( W / V ) methylene blue . Representative images and quantifications of the surviving colony numbers are shown . Data information : Data are shown as mean ± SE from three biological repeats G ) . Statistics : one - way ANOVA ( G ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell survival analysis of the indicated HeLa cells. 7 × 105 cells in one well of 6-well plate were treated with ActD (200 nM) for 4 hours or Doxo (50 nM) for 2 hours and subsequently cultured in normal medium for 21 days. Surviving colonies were stained by 0.1% (W/V) methylene blue. Representative images and quantifications of the surviving colony numbers are shown. Data information: Data are shown as mean ± SE from three biological repeats G). Statistics: one-way ANOVA (G); *P < 0.05, **P < 0.01, ***P < 0.001.


Western blot analysis of BH3 - domain containing proteins in the indicated HeLa cells treated with Doxo ( 10 μM ) . Three proteins with enhanced level in ∆ UBXD8 cells ( Noxa , Bik , and Bnip3 ) are highlighted in red .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of BH3-domain containing proteins in the indicated HeLa cells treated with Doxo (10 μM). Three proteins with enhanced level in ∆UBXD8 cells (Noxa, Bik, and Bnip3) are highlighted in red.


Western blot and quantitative analysis of Noxa degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats ( B , Statistics : two - tailed unpaired Student ' s t - test ( B , ; * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot and quantitative analysis of Noxa degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.001, ***P < 0.001.


Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats C , Statistics : two - tailed unpaired Student ' s t - test C , ; * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 .

Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats C, Statistics: two-tailed unpaired Student's t-test C, ; *P < 0.05, **P < 0.001, ***P < 0.001.


Western blot ( D , analysis of the indicated HeLa cells .

Western blot (D, analysis of the indicated HeLa cells.


Western blot F ) analysis of the indicated HeLa cells .

Western blot F) analysis of the indicated HeLa cells.


Representative images of the mitophagy events in the indicated HeLa cells . mtKeima emission excited by 405 nm laser is shown as green ; mtKeima emission excited by 561 nm laser is shown as red . Mitolysosomes are pointed by white arrows . Scale bar , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of the mitophagy events in the indicated HeLa cells. mtKeima emission excited by 405 nm laser is shown as green; mtKeima emission excited by 561 nm laser is shown as red. Mitolysosomes are pointed by white arrows. Scale bar, 5 μm.


C - F . Quantitative analysis of mitophagy events in the indicated HeLa cells . Only cells with mitophagy were selected to quantify mitolysosome numbers ( D , F ) . Data information : Data are shown as mean ± SE from three biological repeats ( C - F , Statistics : two - tailed unpaired Student ' s t - test ( C - F , ; * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-F. Quantitative analysis of mitophagy events in the indicated HeLa cells. Only cells with mitophagy were selected to quantify mitolysosome numbers (D, F). Data information: Data are shown as mean ± SE from three biological repeats (C-F, Statistics: two-tailed unpaired Student's t-test (C-F, ; **P < 0.01, ***P < 0.001.


I - K , Representative images ( I ) and quantitative analysis of mitophagy events ( J , K ) in the indicated HeLa cells . White arrows point to mitolysosomes . Scale bar , 5 μm . Data information : Data are shown as mean ± SE from three biological repeats J , K , Statistics : two - tailed unpaired Student ' s t - test J , K ) ; * * P < 0 . 01 , * * * P < 0 . 001 .

I-K, Representative images (I) and quantitative analysis of mitophagy events (J, K) in the indicated HeLa cells. White arrows point to mitolysosomes. Scale bar, 5 μm. Data information: Data are shown as mean ± SE from three biological repeats J, K, Statistics: two-tailed unpaired Student's t-test J, K); **P < 0.01, ***P < 0.001.


A . Scatter plot of the relative timing of cell constriction versus the relative timing of nucleoid separation . The gray scale indicates the density of dots in a given area of the chart . The dotted contours represent the 0 . 5 , 0 . 75 and 0 . 95 probability contours of the 240 WT replicates . The Pearson correlation is ρ = 0 . 65 , with a 95 % CI = [ 0 . 63 , 0 . 66 ] . The black dotted diagonal represents the line where a strain should be if both nucleoid separation and cell constriction happens at the same time . Red dots highlight

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Scatter plot of the relative timing of cell constriction versus the relative timing of nucleoid separation. The gray scale indicates the density of dots in a given area of the chart. The dotted contours represent the 0.5, 0.75 and 0.95 probability contours of the 240 WT replicates. The Pearson correlation is ρ = 0.65, with a 95% CI = [0.63, 0.66]. The black dotted diagonal represents the line where a strain should be if both nucleoid separation and cell constriction happens at the same time. Red dots highlight strains


C . Average dynamics of nucleoid separation and cell constriction for WT and the ΔmatP mutant strain . The cumulative distributions of the fraction of cells with two nucleoids ( blue ) and of the fraction of cells with a constriction degree above 0 . 15 ( red ) were plotted against cell age . Cell age was calculated according to the rank of each cell based on their cell length with the formula $ \ text { age } _ { i } \ left ( F \ right ) = \ - \ frac { ln ( 1 - \ frac { F } { 2 } ) } { ln ( 2 ) } $ , where F represents the fraction of cells with a cell length equal or below the length of cell i ( Wold et al , 1994 ) . D . Same plots as in C for three strains clustering in island 8 with the ΔmatP strain . The WT curves shown in C were plotted in gray for comparison . E . Same plots as in C for two island - 8 strains , ΔycjV and ΔhslU , and the ΔhslV strain , which does not partition in island 8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Average dynamics of nucleoid separation and cell constriction for WT and the ΔmatP mutant strain. The cumulative distributions of the fraction of cells with two nucleoids (blue) and of the fraction of cells with a constriction degree above 0.15 (red) were plotted against cell age. Cell age was calculated according to the rank of each cell based on their cell length with the formula $\text{age}_{i}\left( F \right) = \ - \frac{ln(1 - \frac{F}{2})}{ln(2)}$, where F represents the fraction of cells with a cell length equal or below the length of cell i (Wold et al, 1994). D. Same plots as in C for three strains clustering in island 8 with the ΔmatP strain. The WT curves shown in C were plotted in gray for comparison. E. Same plots as in C for two island-8 strains, ΔycjV and ΔhslU, and the ΔhslV strain, which does not partition in island 8.


B . The cumulative distribution of the proportion of constricting cells for the ftsA * mutant and its parent were plotted against cell age . We measured the degree of constriction of all cells using Oufti . For each strain , we had two independently acquired image sets ( n = 496 and 1 , 168 cells for the WT replicates and n = 692 and 1 , 506 cells for the ftsA * replicates ) . The distributions of the degree of constriction were not significantly different among the four datasets at a threshold p - value of 0 . 01 using a Kruskal - Wallis multi - comparison test . Moreover , Bonferroni - corrected post - hoc pairwise tests did not allow a distinction between WT and ftsA * samples .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. The cumulative distribution of the proportion of constricting cells for the ftsA* mutant and its parent were plotted against cell age. We measured the degree of constriction of all cells using Oufti. For each strain, we had two independently acquired image sets (n = 496 and 1,168 cells for the WT replicates and n = 692 and 1,506 cells for the ftsA* replicates). The distributions of the degree of constriction were not significantly different among the four datasets at a threshold p-value of 0.01 using a Kruskal-Wallis multi-comparison test. Moreover, Bonferroni-corrected post-hoc pairwise tests did not allow a distinction between WT and ftsA* samples.


C . Scatter plot of the CV of the cell length for the whole population versus the CV of the cell length for constricting cells only . The contour lines represent the 0 . 5 , 075 and 0 . 95 probability envelopes of the 240 independent WT replicates . The gray color levels indicate the density of points in the vicinity of each strain . The ∆ mraZ strain discussed in the text is highlighted in red .

C. Scatter plot of the CV of the cell length for the whole population versus the CV of the cell length for constricting cells only. The contour lines represent the 0.5, 075 and 0.95 probability envelopes of the 240 independent WT replicates. The gray color levels indicate the density of points in the vicinity of each strain. The ∆mraZ strain discussed in the text is highlighted in red.


( A ) Western blotting of AXL and OPCML protein levels in AXL - expressing , OPCML - null SKOV3 and PEA1 cell lines transduced with OPCML " O " or control Empty vector " E "

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Western blotting of AXL and OPCML protein levels in AXL-expressing, OPCML-null SKOV3 and PEA1 cell lines transduced with OPCML "O" or control Empty vector "E"


( B ) Mammalian 2 - Hybrid assay between OPCML and AXL Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Mammalian 2-Hybrid assay between OPCML and AXL Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


( C ) Western blotting of the OPCML - GST pull down . Input : 1 / 20 of SKOV3 - Empty whole cell

O O O O O O O O O O O O O O O O O O O O O O O O

(C) Western blotting of the OPCML-GST pull down. Input: 1/20 of SKOV3-Empty whole cell lysate


( D ) Western blotting of the anti - AXL immunoprecipitation . Input : 1 / 100 of SKOV3 - OPCML whole cell lysate . IP : Immunoprecipitated protein , IB : Immunoblotted

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Western blotting of the anti-AXL immunoprecipitation. Input: 1/100 of SKOV3-OPCML whole cell lysate. IP: Immunoprecipitated protein, IB: Immunoblotted protein


( E ) Immunostaining of OPCML ( red ) and AXL ( green ) in SKOV3 - OPCML and PEA1 - OPCML cells . Scale bar = 10μm Data i M , ( E ) was performed once due to the limited numbers of primary cells ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Immunostaining of OPCML (red) and AXL (green) in SKOV3-OPCML and PEA1-OPCML cells. Scale bar = 10μm Data i M, (E) was performed once due to the limited numbers of primary cells;


( G ) FRET efficiency for AXL - OPCML interaction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " , using AXL - AXL FRET as a positive control . Cells were labeled with Anti - Axl rabbit ( conjugated to donor probe ) and Anti - OPCML mouse ( conjugated to acceptor probe ) , or Anti - Axl mouse ( conjugated to acceptor probe ) and Anti - Axl rabbit ( conjugated to donor probe ) Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) FRET efficiency for AXL-OPCML interaction in SKOV3-Empty "E" and SKOV3-OPCML "O", using AXL-AXL FRET as a positive control. Cells were labeled with Anti-Axl rabbit (conjugated to donor probe) and Anti-OPCML mouse (conjugated to acceptor probe), or Anti-Axl mouse (conjugated to acceptor probe) and Anti-Axl rabbit (conjugated to donor probe) Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


( H ) PLA of AXL - OPCML ( red ) in SKOV3 and PEA1 cells transduced with empty vector " E " or OPCML " O " . Scale bar =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) PLA of AXL-OPCML (red) in SKOV3 and PEA1 cells transduced with empty vector "E" or OPCML "O". Scale bar = 50μm


Primary ovarian tumour cells were characterised for AXL and OPCML levels by ( I ) Western blottin PAX8 was used to identify ovarian cancer

Primary ovarian tumour cells were characterised for AXL and OPCML levels by (I) Western blottin PAX8 was used to identify ovarian cancer cells


Primary ovarian tumour cells were characterised for AXL and OPCML levels b ( J ) Immunostaining . PAX8 was used to identify ovarian cancer

Primary ovarian tumour cells were characterised for AXL and OPCML levels b (J) Immunostaining. PAX8 was used to identify ovarian cancer cells


( K ) Quantitation of AXL - OPCML PLA in primary cells Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t

(K) Quantitation of AXL-OPCML PLA in primary cells Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


( L ) Western blotting of membrane fractionation of SKOV3 - Empty cells : total cell lysate " T " , liquid - disordered soluble fraction " S " , detergent resistant membrane fraction " R " . Caveolin - 1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction , E : SKOV3 - Empty , O : SKOV3 -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) Western blotting of membrane fractionation of SKOV3-Empty cells: total cell lysate "T", liquid-disordered soluble fraction "S", detergent resistant membrane fraction "R". Caveolin-1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction, E: SKOV3-Empty, O: SKOV3-OPCML


( M ) Densitometry of AXL band intensities in ( J ) in S and R , normalized to AXL intensity in input . Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t

(M) Densitometry of AXL band intensities in (J) in S and R, normalized to AXL intensity in input. Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


SKOV3 - OPCML cells stimulated with Gas6 over a 12 hour time course and ( A ) stained with DAPI ( cyan ) , Anti - OPCML ( green ) and Anti - AXL ( red ) antibodies ( scale bar = 10μm )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SKOV3-OPCML cells stimulated with Gas6 over a 12 hour time course and (A) stained with DAPI (cyan), Anti-OPCML (green) and Anti-AXL (red) antibodies (scale bar = 10μm)


( C , D ) OPCML - AXL interaction ( red ) was visualised by PLA ( scale bar = 50μm ) and quantified , n = 3 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

(C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


d ; ( C , D ) OPCML - AXL interaction ( red ) was visualised by PLA ( scale bar = 50μm ) and quantified , n =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

d; (C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3


( E ) Quantitation OPCML - AXL PLA interaction in primary ovarian tumour cells , n = 1 Data i M , ( E ) was performed once due to the limited numbers of primary cells ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Quantitation OPCML-AXL PLA interaction in primary ovarian tumour cells, n=1 Data i M, (E) was performed once due to the limited numbers of primary cells; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( F ) Co - immunoprecipitation with anti - AXL antibody in cells stimulated with Gas6 as indicated . Input : 1 / 50 of whole cell lysate from SKOV3 - OPCML cells . IP : Immunoprecipitated protein , IB : Immunoblotted

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Co-immunoprecipitation with anti-AXL antibody in cells stimulated with Gas6 as indicated. Input: 1/50 of whole cell lysate from SKOV3-OPCML cells. IP: Immunoprecipitated protein, IB: Immunoblotted protein


( G ) Co - immunoprecipitation with anti - pAXL antibody in SKOV3 - OPCML cells stimulated with

(G) Co-immunoprecipitation with anti-pAXL antibody in SKOV3-OPCML cells stimulated with Gas6


( H ) OPCML - GST pull down assay in SKOV3 - Empty cells stimulated with

(H) OPCML-GST pull down assay in SKOV3-Empty cells stimulated with Gas6


( I ) Membrane fractionation in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells into the detergent resistant " R " membrane fraction and the detergent soluble " S " fraction upon Gas6

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Membrane fractionation in SKOV3-Empty "E" and SKOV3-OPCML "O" cells into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation


( K ) FRAP recovery curves in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) FRAP recovery curves in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( L ) Diffusion Co - efficient in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) Diffusion Co-efficient in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( M ) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

(M) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( N ) FRET Efficiency in SKOV3 - OPCML " O " cells stimulated with Gas6 , and labeled with Anti - Axl ( donor probe ) and Anti - OPCML ( acceptor probe ) Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N) FRET Efficiency in SKOV3-OPCML "O" cells stimulated with Gas6, and labeled with Anti-Axl (donor probe) and Anti-OPCML (acceptor probe) Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests


( O ) Western blotting of the " S " and " R " membrane fractions in SKOV3 - Empty " E " and OCPML " O " cells stimulated with Gas6 for 3 hours , for Gas6 , AXL and pAXL . Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(O) Western blotting of the "S" and "R" membrane fractions in SKOV3-Empty "E" and OCPML "O" cells stimulated with Gas6 for 3 hours, for Gas6, AXL and pAXL. Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM


( A ) Filipin staining of SKOV3 - OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0 , 30 minutes , and 12 hours in serum free ( SF )

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(A) Filipin staining of SKOV3-OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0, 30 minutes, and 12 hours in serum free (SF) medium


( B ) Western blotting of SKOV3 - OPCML cells treated with MBCD and separated into the detergent resistant " R " membrane fraction and the detergent soluble " S " fraction upon Gas6

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(B) Western blotting of SKOV3-OPCML cells treated with MBCD and separated into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation


( C ) Ratio of AXL band intensity in " R " or pAXL band intensity in " S " relative to total AXL " T " from SKOV3 - OPCML " O " in ( B )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Ratio of AXL band intensity in "R" or pAXL band intensity in "S" relative to total AXL "T" from SKOV3-OPCML "O" in (B)


( D ) MBCD treatment of SKOV3 - EMPTY and SKOV3 - OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot . GAPDH was used as loading

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) MBCD treatment of SKOV3-EMPTY and SKOV3-OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot. GAPDH was used as loading control


( E , F ) Signalling kinetics are presented for pAXL and pERK in SKOV3 - EMPTY " E " and SKOV3 - OCPML " O " , treated with MBCD and no treatment control , normalised to GAPDH , from ( D )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(E,F) Signalling kinetics are presented for pAXL and pERK in SKOV3-EMPTY "E" and SKOV3-OCPML "O", treated with MBCD and no treatment control, normalised to GAPDH, from (D)


( G ) PLA assay of AXL - OPCML interaction ( red ) upon Gas6 stimulation in both MBCD treated and non - treated SKOV3 - OPCML cells . Scale bar =

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(G) PLA assay of AXL-OPCML interaction (red) upon Gas6 stimulation in both MBCD treated and non-treated SKOV3-OPCML cells. Scale bar = 50μm


( B ) Western Blotting of SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells pre incubated with either no drug ( DMSO only control ) , 1 . 09 μM R428 , or 2 . 84 μM R428 for an hour and stimulated with Gas6 for the indicated times . GAPDH was used as loading

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(B) Western Blotting of SKOV3-Empty "E" and SKOV3-OPCML "O" cells pre incubated with either no drug (DMSO only control), 1.09 μM R428, or 2.84 μM R428 for an hour and stimulated with Gas6 for the indicated times. GAPDH was used as loading control


( C ) Immunofluorescence staining of pERK ( red ) and OPCML ( magenta ) in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with R428 and stimulated with Gas6 for the indicted time points . Scale bars =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(C) Immunofluorescence staining of pERK (red) and OPCML (magenta) in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with R428 and stimulated with Gas6 for the indicted time points. Scale bars = 50μm


D Western blot of Sec63 , mCherry and Pgk1 from WT cells ( SSY1404 ) , cells harboring the estradiol - inducible system ( SSY1405 ) or ∆ opi1 cells ( SSY1607 ) , all of which expressed Sec63 - mNeon and Rtn1 - mCherry . Cells were untreated or treated with 800 nM estradiol for 6 h . Pgk1 served as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Western blot of Sec63, mCherry and Pgk1 from WT cells (SSY1404), cells harboring the estradiol-inducible system (SSY1405) or ∆opi1 cells (SSY1607), all of which expressed Sec63-mNeon and Rtn1-mCherry. Cells were untreated or treated with 800 nM estradiol for 6 h. Pgk1 served as a loading control.


E Quantification of ER size in estradiol - treated cells harboring the inducible system ( SSY1405 ) , untreated ∆ opi1 cells ( SSY1607 ) and WT cells ( SSY1404 ) treated with 8 mM DTT for 1 h . Bars represent mean + s . e . m . , n = 3 biological replicates . Asterisks indicate statistical significance compared with 0 nM estradiol or ∆ opi1 cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * , P < 0 . 05 ; * * , P < 0 . 01 ; n . s . , not significant .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Quantification of ER size in estradiol-treated cells harboring the inducible system (SSY1405), untreated ∆opi1 cells (SSY1607) and WT cells (SSY1404) treated with 8 mM DTT for 1 h. Bars represent mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with 0 nM estradiol or ∆opi1 cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.


A Sec63 - mNeon images of cells of the indicated genotypes harboring the inducible system . Cells were treated with 800 nM estradiol for 6 h . Two examples of each phenotypic class are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Sec63-mNeon images of cells of the indicated genotypes harboring the inducible system. Cells were treated with 800 nM estradiol for 6 h. Two examples of each phenotypic class are shown.


C Sec63 - mNeon and Rtn1 - mCherry images of ∆ lnp1 and ∆ sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Sec63-mNeon and Rtn1-mCherry images of ∆lnp1 and ∆sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h.


C Quantification of peripheral ER structures in WT and ∆ ice2 cells harboring the inducible system ( SSY1405 , 1603 ) and treated with 800 nM estradiol for the times indicated . Bars are the mean percentage of cell cortex covered by tubules ( purple ) or sheets ( green ) , n = 3 biological replicates . Upper error bars are s . e . m . for the sum of tubules and sheets , lower error bars are s . e . m . for sheets . Asterisks indicate statistical significance compared with the corresponding value in WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 ; n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Quantification of peripheral ER structures in WT and ∆ice2 cells harboring the inducible system (SSY1405, 1603) and treated with 800 nM estradiol for the times indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.


E Quantification of peripheral ER structures in untreated WT , Δice2 , Δopi1 and Δice2 Δopi1 cells ( SSY1404 , 2356 , 2595 , 2811 ) . Bars are the mean percentage of cell cortex covered by tubules ( purple ) or sheets ( green ) , n = 3 biological replicates . Upper error bars are s . e . m . for the sum of tubules and sheets , lower error bars are s . e . m . for sheets . Asterisks indicate statistical significance compared with the corresponding value in WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 ; n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Quantification of peripheral ER structures in untreated WT, Δice2, Δopi1 and Δice2 Δopi1 cells (SSY1404, 2356, 2595, 2811). Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.


C , D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63 - mNeon and Rtn1 - mCherry ( SSY1405 , 1603 ) that were untreated ( C ) or treated with 8 mM DTT for 1 h ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

C, D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63-mNeon and Rtn1-mCherry (SSY1405, 1603) that were untreated (C) or treated with 8 mM DTT for 1 h (D).


B Growth assays of untreated WT , Δice2 , Δnem1 , Δnem1 Δice2 , Δspo7 , Δspo7 Δice2 , Δpah1 and Δpah1 Δice2 cells ( SSY1404 , 2356 , 2482 , 2484 , 2481 , 2483 , 2807 , 2808 ) . Numbers represent areas under the curves and serve as growth indices . Mean + s . e . m . , n = 3 biological replicates . Data for WT and ∆ ice2 cells are the same in the left and middle panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Growth assays of untreated WT, Δice2, Δnem1, Δnem1 Δice2, Δspo7, Δspo7 Δice2, Δpah1 and Δpah1 Δice2 cells (SSY1404, 2356, 2482, 2484, 2481, 2483, 2807, 2808). Numbers represent areas under the curves and serve as growth indices. Mean + s.e.m., n = 3 biological replicates. Data for WT and ∆ice2 cells are the same in the left and middle panels.


C , D Lipidomic analysis of WT , Δice2 , Δnem1 , Δice2 Δnem1 , Δspo7 and Δice2 Δspo7 cells ( SSY1404 , 2356 , 2482 , 2484 , 2481 , 2483 ) . Mean + s . e . m . , n = 4 biological replicates . Asterisks indicate statistical significance compared with WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * , P < 0 . 05 ; * * , P < 0 . 01 . Data for WT and Δice2 cells are the same as in both panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D Lipidomic analysis of WT, Δice2, Δnem1, Δice2 Δnem1, Δspo7 and Δice2 Δspo7 cells (SSY1404, 2356, 2482, 2484, 2481, 2483). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01. Data for WT and Δice2 cells are the same as in both panels.


E Sec63-mNeon images of untreated WT, Δnem1, Δnem1Δice2, Δspo7 and Δspo7 Δice2 cells (SSY1404, 2482, 2484, 2481, 2483).


D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1 - HA from ∆ nem1 mutants ( SSY3065 ) and microsomes from cells of the indicated genotypes ( SSY3053 , 3074 , 3075 , 3095 ) . Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase ( PPase ) are shown for reference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1-HA from ∆nem1 mutants (SSY3065) and microsomes from cells of the indicated genotypes (SSY3053, 3074, 3075, 3095). Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase (PPase) are shown for reference.


E Western blot of HA , Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ ice2 cells expressing Nem1 - HA ( SSY3140 , 3141 ) . Nem1 is undetectable in cell lysates due to its low abundance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Western blot of HA, Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ice2 cells expressing Nem1-HA (SSY3140, 3141). Nem1 is undetectable in cell lysates due to its low abundance.