5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . qPCR of Ucp1 levels in inguinal white adipose tissue ( iWAT ) 5 weeks post injection ( n = 3 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis .

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5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. qPCR of Ucp1 levels in inguinal white adipose tissue (iWAT) 5 weeks post injection (n=3 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.


5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Western blot analysis ( H ) of Ucp1 levels in inguinal white adipose tissue ( iWAT ) 5 weeks post injection ( n = 3 per group , 2 independent repeats ) . Hsp90 , loading control for Western blot analysis .

O O O O O B-CONTROLLED_VAR 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 B-MEASURED_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

5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Western blot analysis (H) of Ucp1 levels in inguinal white adipose tissue (iWAT) 5 weeks post injection (n=3 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.


Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during fasting - feeding Western blot analysis of hepatic Sel1L - Hrd1 ERAD and Crebh of nuclear ( Nuc ) and cytosolic ( Cyto ) fractions from 10 - week - old Sel1Lf / f and Sel1LAlb mice under overnight fasted or fed states .

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Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from 10-week-old Sel1Lf/f and Sel1LAlb mice under overnight fasted or fed states.


Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during fasting - feeding Quantitation of levels of Sel1L / Hrd1 / Crebh proteins and Fgf21 mRNA in the livers of 10 - week - old Sel1Lf / f mice .

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Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA in the livers of 10-week-old Sel1Lf/f mice.


during fasting - feeding Quantitation of total Crebh ( Crebh + Crebh - N ) protein levels in the livers of 10 - week - old Sel1Lf / f and Sel1LAlb mice .

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during fasting-feeding Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers of 10-week-old Sel1Lf/f and Sel1LAlb mice.


during fasting - feeding Serum Fgf21 levels in 10 - week - old Sel1Lf / f and Sel1LAlb mice .

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during fasting-feeding Serum Fgf21 levels in 10-week-old Sel1Lf/f and Sel1LAlb mice.


Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during growth Western blot analysis of hepatic Sel1L - Hrd1 ERAD and Crebh of nuclear ( Nuc ) and cytosolic ( Cyto ) fractions from the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age .

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Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during growth Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.


Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during growth Quantitation of levels of Sel1L / Hrd1 / Crebh proteins and Fgf21 mRNA with the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age .

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Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during growth Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA with the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.


Quantitation of total Crebh ( Crebh + Crebh - N ) protein levels in the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age .

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Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.


Serum Fgf21 levels in the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age .

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Serum Fgf21 levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.


( b ) Upper panel : Representative images of BOEC colonies from PBMC cultivation 10 days post - seeding . Dotted line in each image outlines a BOEC colony . Lower panel : Stabilized BOEC cultures after passaging of colonies ( scale bar , 250 µm ) . ( c ) Proliferation doubling time for NAFLD and healthy BOECs based on passages 2 and 3 . Sample sizes are n = 9 healthy , n = 7 NAFLD ; with 3 biological replicates per donor . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , not significant ( t - test ) .

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(b) Upper panel: Representative images of BOEC colonies from PBMC cultivation 10 days post-seeding. Dotted line in each image outlines a BOEC colony. Lower panel: Stabilized BOEC cultures after passaging of colonies (scale bar, 250 µm). (c) Proliferation doubling time for NAFLD and healthy BOECs based on passages 2 and 3. Sample sizes are n = 9 healthy, n = 7 NAFLD; with 3 biological replicates per donor. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, not significant (t-test).


( d ) Left panel : Flow cytometry characterization of BOECs for endothelial , leukocyte and progenitor cell markers ( grey - isotype control ; red / blue / green / purple - cell lineage marker staining ) . HUVEC and monocytic THP - 1 cells were used as positive controls for endothelial and leukocytic markers respectively . Right panel : Percentages of positively expressing cells . Sample sizes are n = 4 - 6 healthy , n = 2 - 7 NAFLD .

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(d) Left panel: Flow cytometry characterization of BOECs for endothelial, leukocyte and progenitor cell markers (grey - isotype control; red/blue/green/purple - cell lineage marker staining). HUVEC and monocytic THP-1 cells were used as positive controls for endothelial and leukocytic markers respectively. Right panel: Percentages of positively expressing cells. Sample sizes are n = 4-6 healthy, n = 2-7 NAFLD.


( e ) Representative images of immunostaining for endothelial markers , PECAM1 and CDH5 ( scale bar , 100 µm ) .

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(e) Representative images of immunostaining for endothelial markers, PECAM1 and CDH5 (scale bar, 100 µm).


( f ) Left panel : Longitudinal monitoring of BOEC angiogenesis in fibrin gel bead - based sprouting assays over 24h ( representative images ; scale bar , 100 µm ) . Right panel : Box - whisker plots of quantifications of sprout lengths and numbers of sprout per bead at 24h . n = 3 healthy , n = 3 NAFLD , with 4 - 5 beads analyzed per donor and 5 - 11 sprouts per bead .

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(f) Left panel: Longitudinal monitoring of BOEC angiogenesis in fibrin gel bead-based sprouting assays over 24h (representative images; scale bar, 100 µm). Right panel: Box-whisker plots of quantifications of sprout lengths and numbers of sprout per bead at 24h. n = 3 healthy, n = 3 NAFLD, with 4-5 beads analyzed per donor and 5-11 sprouts per bead.


( b ) Volcano plot depicting differentially expressed genes comparing NAFLD BOECs with healthy BOECs . Red and blue dots represent upregulated and downregulated genes in NAFLD BOECs , respectively .

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(b) Volcano plot depicting differentially expressed genes comparing NAFLD BOECs with healthy BOECs. Red and blue dots represent upregulated and downregulated genes in NAFLD BOECs, respectively.


( e ) Heatmap featuring the genes in MCODE Cluster 1 .

(e) Heatmap featuring the genes in MCODE Cluster 1.


( a ) Fold change in gene expressions of CXCL10 , CXCL11 , CXCL12 , CCL20 and CX3CL1 in NAFLD ( n = 10 ) BOECs , normalized to healthy ( n = 12 ) BOECs , with 2 technical replicates per donor . ( c ) Fold change in gene expressions of CXCL10 , CXCL11 , CXCL12 , CCL20 and CX3CL1 in LPS + FFA - treated or non - treated NAFLD ( n = 5 ) and healthy ( n = 5 ) BOECs , normalized to non - treated healthy BOECs , with 3 technical replicates per donor .

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(a) Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20 and CX3CL1 in NAFLD (n = 10) BOECs, normalized to healthy (n = 12) BOECs, with 2 technical replicates per donor. (c) Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20 and CX3CL1 in LPS+FFA-treated or non-treated NAFLD (n = 5) and healthy (n = 5) BOECs, normalized to non-treated healthy BOECs, with 3 technical replicates per donor.


( b ) Left : Representative images showing non - treated and LPS + FFA - treated NAFLD and healthy BOECs , stained with Nile Red ( scale bars , 100 µm ) . Right : Fluorescence intensity of intracellular Nile Red stain was quantified at 515 / 585nm , and readings were normalized with blanks ( n = 3 donors / group ) .

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(b) Left: Representative images showing non-treated and LPS+FFA-treated NAFLD and healthy BOECs, stained with Nile Red (scale bars, 100 µm). Right: Fluorescence intensity of intracellular Nile Red stain was quantified at 515/585nm, and readings were normalized with blanks (n = 3 donors/ group).


( d ) Protein concentrations of chemokines in the conditioned culture media of healthy and NAFLD BOECs . *

(d) Protein concentrations of chemokines in the conditioned culture media of healthy and NAFLD BOECs. *


( e ) Left : Representative images ( Bright field ; DAPI , nuclei ; CDH5 , endothelial cells ; CXCL12 ) of liver sections from chow diet - fed and HFHC diet - fed humanized mice ( scale bar , 40 μm ) . Boxed regions were further magnified ( scale bar , 30 µm ) . Right : Box - whisker plot of Mander ' s Coefficient ( M2 ) that was a measure of the degree of CXCL12 overlap with CDH5 - positive vasculatures in the livers . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . Chow diet - fed ( n = 4 ) and HFHC diet - fed ( n = 4 ) humanized mice were analysed , with 10 - 20 independent regions of interest per mouse were analysed for image quantification .

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(e) Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of liver sections from chow diet-fed and HFHC diet-fed humanized mice (scale bar, 40 μm). Boxed regions were further magnified (scale bar, 30 µm). Right: Box-whisker plot of Mander's Coefficient (M2) that was a measure of the degree of CXCL12 overlap with CDH5-positive vasculatures in the livers. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). Chow diet-fed (n = 4) and HFHC diet-fed (n = 4) humanized mice were analysed, with 10-20 independent regions of interest per mouse were analysed for image quantification.


( f ) Left : Representative images ( Bright field ; DAPI , nuclei ; CDH5 , endothelial cells ; CXCL12 ) of aortic sections from chow diet - fed and LIDPAD mice ( scale bar , 40 μm ) . Boxed regions were further magnified ( scale bar , 30 µm ) . Arrows point at aortic endothelial cells . AW : Aortic wall ; L : lumen . Right : Box - whisker plot of CXCL12 fluorescent intensity in the aortic vascular endothelia . CXCL12 fluorescent intensity for each delimitated endothelial region is represented as individual data points on the plot . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . Chow diet - fed ( n = 3 ) and LIDPAD diet - fed ( n = 3 ) mice were analysed , with 10 - 20 delimitated endothelial regions per mouse analysed for image quantification .

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(f) Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of aortic sections from chow diet-fed and LIDPAD mice (scale bar, 40 μm). Boxed regions were further magnified (scale bar, 30 µm). Arrows point at aortic endothelial cells. AW: Aortic wall; L: lumen. Right: Box-whisker plot of CXCL12 fluorescent intensity in the aortic vascular endothelia. CXCL12 fluorescent intensity for each delimitated endothelial region is represented as individual data points on the plot. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). Chow diet-fed (n = 3) and LIDPAD diet-fed (n = 3) mice were analysed, with 10-20 delimitated endothelial regions per mouse analysed for image quantification.


( g ) Left : Representative images showing aortic sections harvested from mice fed with either chow or LIDPAD diet . Sections were stained for leukocyte markers CD45 ( red ) to determine the extent of leukocyte infiltration . Dotted lines demarcate the aortic walls ( AW ) . L : lumen . Scale bar : 20 µm . Right : Box - whisker plot of mean fluorescence intensity ( MFI ) of CD45 signals as a readout for the amount of infiltrated CD45 + leukocytes within the aortic walls . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . n = 3 mice per group , with 2 tissue sections analyzed per animal .

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(g) Left: Representative images showing aortic sections harvested from mice fed with either chow or LIDPAD diet. Sections were stained for leukocyte markers CD45 (red) to determine the extent of leukocyte infiltration. Dotted lines demarcate the aortic walls (AW). L: lumen. Scale bar: 20 µm. Right: Box-whisker plot of mean fluorescence intensity (MFI) of CD45 signals as a readout for the amount of infiltrated CD45+ leukocytes within the aortic walls. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). n = 3 mice per group, with 2 tissue sections analyzed per animal.


( a ) Upper panel : Single - cell UMAP of sequenced PBMCs from 2 NAFLD patients and 4 healthy subjects ; n = 10 , 786 cells . We identified 15 distinct cell types by DICE annotation . Lower panel : Expression patterns of CXCR3 , CXCR4 , ACKR3 , CX3CR1 , CCR6 across different cell types in NAFLD and healthy PBMC samples .

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(a) Upper panel: Single-cell UMAP of sequenced PBMCs from 2 NAFLD patients and 4 healthy subjects; n = 10,786 cells. We identified 15 distinct cell types by DICE annotation. Lower panel: Expression patterns of CXCR3, CXCR4, ACKR3, CX3CR1, CCR6 across different cell types in NAFLD and healthy PBMC samples.


( c ) Graphic shows the percentage composition of identified cell types in NAFLD and healthy PBMCs single - cell analysis .

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(c) Graphic shows the percentage composition of identified cell types in NAFLD and healthy PBMCs single-cell analysis.


( d ) Upper panel : Representative flow cytometry dot plots , characterizing the isolated CD8 + T cells and CD8 - immune cell fraction ( mainly CD4 + T cells , NK cells and monocytes ) from PBMCs of healthy and NAFLD subjects . Lower panel : Flow cytometry quantification of % of positively expressing CXCR4 + and CX3CR1 + cells within each immune subset . Sample sizes are n = 14 NAFLD ; n = 6 healthy .

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(d) Upper panel: Representative flow cytometry dot plots, characterizing the isolated CD8+ T cells and CD8- immune cell fraction (mainly CD4+ T cells, NK cells and monocytes) from PBMCs of healthy and NAFLD subjects. Lower panel: Flow cytometry quantification of % of positively expressing CXCR4+ and CX3CR1+ cells within each immune subset. Sample sizes are n = 14 NAFLD; n = 6 healthy.


( e ) Heatmap of gene expressions of endothelial antigens relevant to interaction with T lymphocytes in NAFLD ( n = 3 ) and healthy ( n = 3 ) BOECs , with 3 biological replicates per donor .

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(e) Heatmap of gene expressions of endothelial antigens relevant to interaction with T lymphocytes in NAFLD (n = 3) and healthy (n = 3) BOECs, with 3 biological replicates per donor.


( f ) Upper panel : Representative flow cytometry histograms of HLA - A / B / C , HLA - DM and IFN - γR1 expressions in NAFLD ( red ) and healthy ( blue ) BOECs . Grey , isotype control . Lower panel : Box - whisker plots reflect mean fluorescence intensity ( a . u . , arbituary unit ) , normalized to isotype control , as a readout for epitope density . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) . n = 4 donors ; * p < 0 . 05 ; ns , not significant ( Mann - Whitney test for HLA - A / B / C and IFN - γR1 ; t - test for HLA - DM ) .

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(f) Upper panel: Representative flow cytometry histograms of HLA-A/B/C, HLA-DM and IFN-γR1 expressions in NAFLD (red) and healthy (blue) BOECs. Grey, isotype control. Lower panel: Box-whisker plots reflect mean fluorescence intensity (a.u., arbituary unit), normalized to isotype control, as a readout for epitope density. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). n = 4 donors; *p<0.05; ns, not significant (Mann-Whitney test for HLA-A/B/C and IFN-γR1; t-test for HLA-DM).


( a ) Left : Workflow schematic of adhesion assay . Middle : Representative images of labeled THP - 1 and Jurkat cells attached to BOEC monolayers ( Scale bars , 100 µm ) . Right : Box - whisker plots of the numbers of adhered leukocytes were determined fluorometrically . Sample sizes are n = 9 NAFLD and n = 7 healthy , with 3 biological replicates per donor .

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(a) Left: Workflow schematic of adhesion assay. Middle: Representative images of labeled THP-1 and Jurkat cells attached to BOEC monolayers (Scale bars, 100 µm). Right: Box-whisker plots of the numbers of adhered leukocytes were determined fluorometrically. Sample sizes are n = 9 NAFLD and n = 7 healthy, with 3 biological replicates per donor.


( b ) Left : Schematic of chemotaxis assay . Right : Box - whisker plots of the numbers of recruited THP - 1 and Jurkat cells by the BOECs . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) .

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(b) Left: Schematic of chemotaxis assay. Right: Box-whisker plots of the numbers of recruited THP-1 and Jurkat cells by the BOECs. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers).


( c ) CD8 + T cells and CD8 - cell fractions were isolated from NAFLD and healthy subjects . Box - whisker plots show the number of recruited immune cells to their respective BOECs matched by health condition in coculture chemotaxis assays . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) . Sample sizes are n = 11 NAFLD and n = 6 healthy , with 3 - 4 technical replicates per donor .

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(c) CD8+ T cells and CD8- cell fractions were isolated from NAFLD and healthy subjects. Box-whisker plots show the number of recruited immune cells to their respective BOECs matched by health condition in coculture chemotaxis assays. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 11 NAFLD and n = 6 healthy, with 3-4 technical replicates per donor.


( d ) Box - whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with AMD3100 ( CXCR4 small molecule inhibitor ) . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) . Sample sizes are n = 6 NAFLD and n = 4 healthy , with 3 - 4 technical replicates per donor .

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(d) Box-whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with AMD3100 (CXCR4 small molecule inhibitor). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 6 NAFLD and n = 4 healthy, with 3-4 technical replicates per donor.


( e ) Box - whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with anti - CXCL12 IgG ( CXCL12 neutralizing antibody ) . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) . Sample sizes are n = 5 NAFLD and n = 3 healthy , with 3 - 4 technical replicates per donor .

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(e) Box-whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with anti-CXCL12 IgG (CXCL12 neutralizing antibody). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 5 NAFLD and n = 3 healthy, with 3-4 technical replicates per donor.


( f ) Left : Schematic of transendothelial migration assay with patient - derived BOECs and immune cells . Right : Transendothelial electric resistance ( TEER ) measurements of BOEC barrier tightness in the presence and absence of PBMCs and / or AMD3100 . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) . Sample size is n = 5 patient samples , with 3 technical TEER readings recorded per sample .

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(f) Left: Schematic of transendothelial migration assay with patient-derived BOECs and immune cells. Right: Transendothelial electric resistance (TEER) measurements of BOEC barrier tightness in the presence and absence of PBMCs and/or AMD3100. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample size is n = 5 patient samples, with 3 technical TEER readings recorded per sample.


( g ) Box - whisker plots of the numbers of circulating endothelial cells per million PBMCs in healthy ( n = 6 ) and NAFLD subjects ( n = 11 ) . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) .

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(g) Box-whisker plots of the numbers of circulating endothelial cells per million PBMCs in healthy (n = 6) and NAFLD subjects (n = 11). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test).


( A ) . A FRET assay was used to measure the difference in melting temperature ( ΔTm ) of ( G4C2 ) 4 RNA or DNA G - Qs in the presence of 2 μM of 138 different small molecules . An increase in ΔTm indicates stabilisation of the G - Q . Small molecules are ranked on the x axis according to their increasing thermal stabilisation of the DNA ( G4C2 ) 4 G - Q . Small molecules that preferentially stabilise RNA over DNA ( G4C2 ) 4 G - Qs reside in the upper part of the scatter plot , above the blue curve . An arbitrary ΔTm threshold of 13 ° C greater than vehicle ( grey line ) and a differential binding to RNA over DNA ( ΔTmRNA - ΔTmDNA ≥ 5 ° C ) were used to select candidate small molecules .

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(A). A FRET assay was used to measure the difference in melting temperature (ΔTm) of (G4C2)4 RNA or DNA G-Qs in the presence of 2 μM of 138 different small molecules. An increase in ΔTm indicates stabilisation of the G-Q. Small molecules are ranked on the x axis according to their increasing thermal stabilisation of the DNA (G4C2)4 G-Q. Small molecules that preferentially stabilise RNA over DNA (G4C2)4 G-Qs reside in the upper part of the scatter plot, above the blue curve. An arbitrary ΔTm threshold of 13 °C greater than vehicle (grey line) and a differential binding to RNA over DNA (ΔTmRNA-ΔTmDNA ≥ 5 °C) were used to select candidate small molecules.


( C ) . FRET dose - response of DB1246 , DB1247 and DB1273 on stabilisation of RNA or DNA ( G4C2 ) 4 G - Qs .

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(C). FRET dose-response of DB1246, DB1247 and DB1273 on stabilisation of RNA or DNA (G4C2)4 G-Qs.


( D ) . Temperature unfold CD spectra for ( G4C2 ) 4 RNA alone ( which shows a characteristic G - Q structure with minima at 237 nm , maxima at 264 nm and no additional signal ) , or in the presence of 2 μM DB1246 , DB1247 or DB1273 . A characteristic induced CD spectrum , in the 350 nm - 550 nm region , is observed only in the presence of each small molecule , confirming that each of these three compounds are binding to ( G4C2 ) 4 RNA G - Qs . Data in A represent mean ± s . d . , n = 1 with 3 technical replica . Data in C represent mean ± s . d . , n = 3 independent experiments . Data in D represent mean ± s . d . , n = 3 independent experiments .

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(D). Temperature unfold CD spectra for (G4C2)4 RNA alone (which shows a characteristic G-Q structure with minima at 237 nm, maxima at 264 nm and no additional signal), or in the presence of 2 μM DB1246, DB1247 or DB1273. A characteristic induced CD spectrum, in the 350 nm - 550 nm region, is observed only in the presence of each small molecule, confirming that each of these three compounds are binding to (G4C2)4 RNA G-Qs. Data in A represent mean ± s.d., n=1 with 3 technical replica. Data in C represent mean ± s.d., n=3 independent experiments. Data in D represent mean ± s.d., n=3 independent experiments.


( A ) Representative images of C9orf72 iPSC - cortical neurons treated with DMSO ( vehicle control ) or 1 µM of DB1246 , DB1247 or DB1273 , for 4 days . RNA foci are shown in red and nuclei ( DAPI ) in blue . Scale bar represents 10 µm .

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(A) Representative images of C9orf72 iPSC-cortical neurons treated with DMSO (vehicle control) or 1 µM of DB1246, DB1247 or DB1273, for 4 days. RNA foci are shown in red and nuclei (DAPI) in blue. Scale bar represents 10 µm.


( B ) Quantification shows RNA foci are significantly reduced by all three compounds in iPSC - cortical neurons and by DB1246 and DB1273 in iPSC - motor neurons . Data are shown as the average percentage of neurons containing RNA foci relative to vehicle ( DMSO ) . N = 3 independent C9orf72 patient lines with 2 - 3 inductions per line and at least 70 neurons counted per induction . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , one sample two - tailed t - test versus normalised control . For cortical neurons , p = 0 . 0124 ( DB1246 ) , p = 0 . 0065 ( DB1247 ) , p = 0 . 0096 ( DB1273 ) . For motor neurons , p = 0 . 0004 ( DB1246 ) , p = 0 . 0030 ( DB1273 ) .

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(B) Quantification shows RNA foci are significantly reduced by all three compounds in iPSC-cortical neurons and by DB1246 and DB1273 in iPSC-motor neurons. Data are shown as the average percentage of neurons containing RNA foci relative to vehicle (DMSO). N=3 independent C9orf72 patient lines with 2-3 inductions per line and at least 70 neurons counted per induction. *p <0.05, **p <0.01, ***p <0.001, one sample two-tailed t-test versus normalised control. For cortical neurons, p=0.0124 (DB1246), p=0.0065 (DB1247), p=0.0096 (DB1273). For motor neurons, p=0.0004 (DB1246), p=0.0030 (DB1273).


( A ) Poly ( GP ) levels were measured by MSD immunoassay in C9orf72 patient iPSC - motor neurons treated for 7 days with 8 - 16 µM of the G4C2 repeat G - Q binding small molecules DB1247 , DB1247 and DB1273 . Treatment with DB1246 or DB1273 leads to a significant reduction in poly ( GP ) levels relative to vehicle - treated controls . Data are shown as the mean and SEM of 3 independent C9orf72 iPSC lines , with 1 - 6 differentiations per line . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , one sample two - tailed t - test versus normalised control . p = 0 . 0068 ( DB1246 , 12 µm ) , p = 0 . 0417 ( DB1246 , 16 µm ) , p = 0 . 0196 ( DB1247 , 8 µm ) , p = 0 . 0002 ( DB1273 , 8 µm ) , p = 0 . 0194 ( DB1273 , 12 µm ) , p = 0 . 0062 ( DB1273 , 16 µm )

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(A) Poly(GP) levels were measured by MSD immunoassay in C9orf72 patient iPSC-motor neurons treated for 7 days with 8-16 µM of the G4C2 repeat G-Q binding small molecules DB1247, DB1247 and DB1273. Treatment with DB1246 or DB1273 leads to a significant reduction in poly(GP) levels relative to vehicle-treated controls. Data are shown as the mean and SEM of 3 independent C9orf72 iPSC lines, with 1-6 differentiations per line. *p<0.05, **p<0.01, *** p<0.001, one sample two-tailed t-test versus normalised control. p=0.0068 (DB1246, 12 µm), p=0.0417 (DB1246, 16 µm), p=0.0196 (DB1247, 8 µm), p=0.0002 (DB1273, 8 µm), p=0.0194 (DB1273, 12 µm), p=0.0062 (DB1273, 16 µm)


( B ) C9orf72 patient iPSC - motor neurons were treated for 7 days with 16 µM of each small molecule and the expression levels of MCM2 and the three C9orf72 transcript variants ( V1 - V3 ) measured by quantitative RT - PCR . Data are shown as the mean and s . d . of 3 independent iPSC - motor neuron lines ( one induction per line ) relative to vehicle ( DMSO ) treated controls . No significant changes in gene expression were observed , one way ANOVA , Dunnett ' s post hoc test , p > 0 . 05 .

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(B) C9orf72 patient iPSC-motor neurons were treated for 7 days with 16 µM of each small molecule and the expression levels of MCM2 and the three C9orf72 transcript variants (V1-V3) measured by quantitative RT-PCR. Data are shown as the mean and s.d. of 3 independent iPSC-motor neuron lines (one induction per line) relative to vehicle (DMSO) treated controls. No significant changes in gene expression were observed, one way ANOVA, Dunnett's post hoc test, p>0.05.


( C ) XTT cell death assay for C9orf72 iPSC - motor neurons treated for 7 days with 0 - 40 µM of DB1246 , DB1247 , DB1273 or cisplatin as a positive control . Data are shown as the mean and SEM of two independent iPSC - motor neuron lines . Toxicity is only observed at the highest dose of 40 µM and not at the penultimate dose of 20 µM .

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(C) XTT cell death assay for C9orf72 iPSC-motor neurons treated for 7 days with 0 - 40 µM of DB1246, DB1247, DB1273 or cisplatin as a positive control. Data are shown as the mean and SEM of two independent iPSC-motor neuron lines. Toxicity is only observed at the highest dose of 40 µM and not at the penultimate dose of 20 µM.


( D ) Drosophila first instar larvae expressing 36 G4C2 repeats were placed on food containing either vehicle or 1 mM DB1273 , and the number reaching the pupal stage of development counted after 7 days . DB1273 treatment ( n = 1200 flies ) significantly improves survival compared to vehicle ( n = 1150 flies ) , p = 0 . 0320 , chi - squared test . Data are shown as proportion reaching the pupal stage , with numbers within each group indicated on the bars . Genotype was w1118 ; ( GGGGCC ) 36 / + ; daGAL4 / + ( daGAL4 > 36R ) .

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(D) Drosophila first instar larvae expressing 36 G4C2 repeats were placed on food containing either vehicle or 1 mM DB1273, and the number reaching the pupal stage of development counted after 7 days. DB1273 treatment (n=1200 flies) significantly improves survival compared to vehicle (n=1150 flies), p=0.0320, chi-squared test. Data are shown as proportion reaching the pupal stage, with numbers within each group indicated on the bars. Genotype was w1118; (GGGGCC)36/+; daGAL4/+ (daGAL4>36R).


( E ) Drosophila first instar larvae ubiquitously expressing 36 G4C2 repeats ( 36R ) were treated with vehicle or 1 mM DB1273 for 5 days , until the third instar stage ( L3 ) , and poly ( GR ) measured by MSD immunoassay . Poly ( GR ) was also measured in control ( w1118 ) larvae that do not express G4C2 repeats . DB1273 treatment significantly reduced poly ( GR ) expression . n = 8 for 36R groups , n = 7 for control group . * * p < 0 . 01 , * * * p < 0 . 001 , one way ANOVA with Dunnett ' s post hoc test . p = 0 . 0001 ( w1118 versus 36R 0 mM larvae ) , p = 0 . 0019 ( 0 mM versus 1 mM 36R larvae ) . Genotypes were w1118 ; ; and w1118 ; ( GGGGCC ) 36 / + ; daGAL4 / + ( daGAL4 > 36R ) .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

(E) Drosophila first instar larvae ubiquitously expressing 36 G4C2 repeats (36R) were treated with vehicle or 1 mM DB1273 for 5 days, until the third instar stage (L3), and poly(GR) measured by MSD immunoassay. Poly(GR) was also measured in control (w1118) larvae that do not express G4C2 repeats. DB1273 treatment significantly reduced poly(GR) expression. n=8 for 36R groups, n=7 for control group. **p<0.01, *** p<0.001, one way ANOVA with Dunnett's post hoc test. p=0.0001 (w1118 versus 36R 0 mM larvae), p=0.0019 (0 mM versus 1 mM 36R larvae). Genotypes were w1118; ; and w1118; (GGGGCC)36/+; daGAL4/+ (daGAL4>36R).


( A ) Coomassie ‐ stained gels showing the results of liposome co ‐ sedimentation assays using Folch liposomes and the indicated proteins . Liposome binding allows the protein to be pelleted . The star indicates a degradation product of the Atg5 - Atg12 conjugate .

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(A) Coomassie‐stained gels showing the results of liposome co‐sedimentation assays using Folch liposomes and the indicated proteins. Liposome binding allows the protein to be pelleted. The star indicates a degradation product of the Atg5-Atg12 conjugate.


( B , D ) Yeast cells of the indicated genotype were transformed with plasmids coding for Atg5 and Atg16 , respectively , and subjected to fractionation experiments . The proteins were detected by anti ‐ Myc western blotting . The presence of the protein in the pellet fraction indicated membrane binding .

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(B, D) Yeast cells of the indicated genotype were transformed with plasmids coding for Atg5 and Atg16, respectively, and subjected to fractionation experiments. The proteins were detected by anti‐Myc western blotting. The presence of the protein in the pellet fraction indicated membrane binding.


( C ) Quantification showing the relative amounts of Atg5 - 9 × Myc and Atg5 - 9 × Myc - Atg12 in the pellet fractions relative to Pex30 . The amount of Atg5 - 9 × Myc and Atg5 - 9 × Myc - Atg12 in atg5Δ cells was set to 1 . See Supplementary Figure 1 for graph based on non ‐ normed data . The quantification is based on three independent experiments and the averages and the standard deviations are shown . The numbers next to the blots indicate the molecular weight in kDa . P , pellet ; S , supernatant . Figure source data can be found with the Supplementary data .

O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR 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

(C) Quantification showing the relative amounts of Atg5-9 × Myc and Atg5-9 × Myc-Atg12 in the pellet fractions relative to Pex30. The amount of Atg5-9 × Myc and Atg5-9 × Myc-Atg12 in atg5Δ cells was set to 1. See Supplementary Figure 1 for graph based on non‐normed data. The quantification is based on three independent experiments and the averages and the standard deviations are shown. The numbers next to the blots indicate the molecular weight in kDa. P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.


( A ) Coomassie ‐ stained gels of liposome co ‐ sedimentation assays using either Atg5 - Atg12 / Atg16 ( upper panel ) or Atg5 / Atg16 ( lower panel ) and liposomes with the indicated lipid composition ( Folch : Folch lipids , PE / PI3P : 40 % POPC , 35 % POPS , 20 % POPE , 5 % PI3P , PE / PI : 40 % POPC , 35 % POPS , 20 % POPE , 5 % PI , DAG / PI3P : 40 % POPC , 35 % POPS , 20 % DAG , 5 % PI3P , DAG / PI : 40 % POPC , 35 % POPS , 20 % DAG , 5 % PI ) . Note that the Atg5 - Atg12 / Atg16 and the Atg5 / Atg16 complexes show almost identical binding behaviours .

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(A) Coomassie‐stained gels of liposome co‐sedimentation assays using either Atg5-Atg12/Atg16 (upper panel) or Atg5/Atg16 (lower panel) and liposomes with the indicated lipid composition (Folch: Folch lipids, PE/PI3P: 40% POPC, 35% POPS, 20% POPE, 5% PI3P, PE/PI: 40% POPC, 35% POPS, 20% POPE, 5% PI, DAG/PI3P: 40% POPC, 35% POPS, 20% DAG, 5% PI3P, DAG/PI: 40% POPC, 35% POPS, 20% DAG, 5% PI). Note that the Atg5-Atg12/Atg16 and the Atg5/Atg16 complexes show almost identical binding behaviours.


( B ) Quantification showing the amounts of Atg5 - Atg12 / Atg16 and Atg5 / Atg16 in the pellet fractions . The amount of protein in the pellet fraction in the absence of liposomes ( background ) was subtracted from the amount of protein in the pellet fractions in the presence of liposomes . Data were obtained from three independent experiments , one of which is shown in ( A ) . The Atg5 - Atg12 and Atg5 bands were used for quantification .

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(B) Quantification showing the amounts of Atg5-Atg12/Atg16 and Atg5/Atg16 in the pellet fractions. The amount of protein in the pellet fraction in the absence of liposomes (background) was subtracted from the amount of protein in the pellet fractions in the presence of liposomes. Data were obtained from three independent experiments, one of which is shown in (A). The Atg5-Atg12 and Atg5 bands were used for quantification.


( C ) Coomassie ‐ stained gel of a liposome co ‐ sedimentation assay showing the lipid requirements of the Atg5 - Atg12 / Atg16 complex . Liposomes were composed of 40 % POPC , 35 % POPS , 20 % POPE and 5 % PI3P . In the liposomes containing no POPE or POPS , these lipids were replaced with POPC .

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(C) Coomassie‐stained gel of a liposome co‐sedimentation assay showing the lipid requirements of the Atg5-Atg12/Atg16 complex. Liposomes were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. In the liposomes containing no POPE or POPS, these lipids were replaced with POPC.


( D ) Quantification of Atg5 - Atg12 / Atg16 in the pellet fraction in liposome co ‐ sedimentation , one of which is shown in ( C ) . Data were obtained from six independent experiments . The Atg5 - Atg12 was used for quantification . The average value is shown and the error bars represent the standard deviation . P ‐ values were calculated using Student ' s t ‐ test . The numbers next to the gels indicate the molecular weight in kDa . DAG , diacylglycerol ; P , pellet ; PI , phosphatidylinositol ; PI3P , phosphatidylinositol ‐ 3 ‐ phosphate ; ( PO ) PC , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylcholine ; ( PO ) PE , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylethanolamine ; ( PO ) PS , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylserine ; S , supernatant . Figure source data can be found with the Supplementary data .

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(D) Quantification of Atg5-Atg12/Atg16 in the pellet fraction in liposome co‐sedimentation, one of which is shown in (C). Data were obtained from six independent experiments. The Atg5-Atg12 was used for quantification. The average value is shown and the error bars represent the standard deviation. P‐values were calculated using Student's t‐test. The numbers next to the gels indicate the molecular weight in kDa. DAG, diacylglycerol; P, pellet; PI, phosphatidylinositol; PI3P, phosphatidylinositol‐3‐phosphate; (PO)PC, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylcholine; (PO)PE, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylethanolamine; (PO)PS, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylserine; S, supernatant. Figure source data can be found with the Supplementary data.


( A ) Coomassie ‐ stained gels showing co ‐ sedimentation assays using Folch liposomes and the indicated proteins . Atg5 - Atg12 / Atg16 was most efficient in recruiting Atg3 to the pellet fraction .

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(A) Coomassie‐stained gels showing co‐sedimentation assays using Folch liposomes and the indicated proteins. Atg5-Atg12/Atg16 was most efficient in recruiting Atg3 to the pellet fraction.


( B ) Quantification of Atg3 in the pellet fraction in the presence of the indicated proteins . The quantification is based on three independent experiments .

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(B) Quantification of Atg3 in the pellet fraction in the presence of the indicated proteins. The quantification is based on three independent experiments.


( C ) Coomassie ‐ stained gels showing the results of Atg8 - PE conjugation assays using the indicated proteins and E . coli lipids derived liposomes . Atg8 - PE conjugation was detected as characteristic band shift as the conjugated form runs faster on urea ‐ SDS gels . The numbers above the gels indicate the time in minutes . Note that at least under these conditions almost no Atg8 - PE conjugation was detected in the absence of the Atg5 - Atg12 conjugate or the Atg5 - Atg12 / Atg16 complex . The numbers next to the gels indicate the molecular weight in kDa . The averages and the error bars representing the standard deviations are shown . P ‐ values were calculated using Student ' s t ‐ test . NS , non ‐ significant ; P , pellet ; S , supernatant . Figure source data can be found with the Supplementary data .

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(C) Coomassie‐stained gels showing the results of Atg8-PE conjugation assays using the indicated proteins and E. coli lipids derived liposomes. Atg8-PE conjugation was detected as characteristic band shift as the conjugated form runs faster on urea‐SDS gels. The numbers above the gels indicate the time in minutes. Note that at least under these conditions almost no Atg8-PE conjugation was detected in the absence of the Atg5-Atg12 conjugate or the Atg5-Atg12/Atg16 complex. The numbers next to the gels indicate the molecular weight in kDa. The averages and the error bars representing the standard deviations are shown. P‐values were calculated using Student's t‐test. NS, non‐significant; P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.


( A ) Confocal microscopy images showing eGFP - Atg8 recruitment to GUVs is dependent on the Atg5 - Atg12 / Atg16 complex and PE . The membrane was labelled by incorporation of rhodamine ‐ PE .

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(A) Confocal microscopy images showing eGFP-Atg8 recruitment to GUVs is dependent on the Atg5-Atg12/Atg16 complex and PE. The membrane was labelled by incorporation of rhodamine‐PE.


( B ) Single confocal section showing colocalization of Atg5 - Atg12 / Atg16 - eGFP and mCherry - Atg8 on GUVs .

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(B) Single confocal section showing colocalization of Atg5-Atg12/Atg16-eGFP and mCherry-Atg8 on GUVs.


( C ) Quantification of GUV ‐ associated eGFP - Atg8 in the presence of the Atg5 - Atg12 / Atg16 complex and the Atg5 - Atg12 conjugate . The first graph shows the percentage of GUVs associated with eGFP - Atg8 fluorescence . The second graph shows the intensity of eGFP - Atg8 on the GUVs . The quantification is based on four independent experiments . Inset : anti ‐ Atg12 western blot using the rabbit anti ‐ Atg12 antiserum showing the amounts of Atg5 - Atg12 / Atg16 complex and Atg5 - Atg12 conjugate in one of the assays used for the quantification shown in ( C ) .

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(C) Quantification of GUV‐associated eGFP-Atg8 in the presence of the Atg5-Atg12/Atg16 complex and the Atg5-Atg12 conjugate. The first graph shows the percentage of GUVs associated with eGFP-Atg8 fluorescence. The second graph shows the intensity of eGFP-Atg8 on the GUVs. The quantification is based on four independent experiments. Inset: anti‐Atg12 western blot using the rabbit anti‐Atg12 antiserum showing the amounts of Atg5-Atg12/Atg16 complex and Atg5-Atg12 conjugate in one of the assays used for the quantification shown in (C).


( D ) Quantification of membrane recruitment of Atg3 - mCherry by the Atg5 - Atg12 / Atg16 complex , the Atg5 - Atg12 conjugate and a buffer control . The graph is based on two independent experiments .

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(D) Quantification of membrane recruitment of Atg3-mCherry by the Atg5-Atg12/Atg16 complex, the Atg5-Atg12 conjugate and a buffer control. The graph is based on two independent experiments.


( E ) Widefield images showing individual frames of a time series of GUVs incubated with Atg3 , Atg7 , Atg8 and the Atg5 - Atg12 / Atg16 complex ( see Materials and methods for details ) . The membrane was labelled by incorporation of rhodamine ‐ PE . The time is indicated in minutes .

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(E) Widefield images showing individual frames of a time series of GUVs incubated with Atg3, Atg7, Atg8 and the Atg5-Atg12/Atg16 complex (see Materials and methods for details). The membrane was labelled by incorporation of rhodamine‐PE. The time is indicated in minutes.


( F ) Widefield images of GUVs incubated with Atg3 , Atg7 , eGFP - Atg8 and the Atg5 - Atg12 / Atg16 complex for 35 min ( left ) or 70 min ( right ) . The membrane was labelled by incorporation of rhodamine ‐ PE . Note the massive accumulation of eGFP - Atg8 at the interface between individual GUVs . The arrows point to some of these interfaces . For the quantifications , the averages and standard deviations are shown . P ‐ values were calculated using Student ' s t ‐ test . Scale bars : 10 μm . Figure source data can be found with the Supplementary data .

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(F) Widefield images of GUVs incubated with Atg3, Atg7, eGFP-Atg8 and the Atg5-Atg12/Atg16 complex for 35 min (left) or 70 min (right). The membrane was labelled by incorporation of rhodamine‐PE. Note the massive accumulation of eGFP-Atg8 at the interface between individual GUVs. The arrows point to some of these interfaces. For the quantifications, the averages and standard deviations are shown. P‐values were calculated using Student's t‐test. Scale bars: 10 μm. Figure source data can be found with the Supplementary data.


( A ) Frames of widefield images taken from a time series of rhodamine ‐ PE labelled GUVs in the presence of the Atg5 - Atg12 / Atg16 complex . For the control condition buffer but no protein was added . Note the increase in clustered GUVs over time . The numbers indicate the time in minutes .

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(A) Frames of widefield images taken from a time series of rhodamine‐PE labelled GUVs in the presence of the Atg5-Atg12/Atg16 complex. For the control condition buffer but no protein was added. Note the increase in clustered GUVs over time. The numbers indicate the time in minutes.


( B ) Confocal images of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP complex . Note the accumulation of the complex at the interface between GUVs ( arrows ) .

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(B) Confocal images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP complex. Note the accumulation of the complex at the interface between GUVs (arrows).


( C ) Individual confocal microscopy frames of a time series of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP . The arrows point to the GUV - GUV interface where the protein initially accumulated .

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(C) Individual confocal microscopy frames of a time series of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the GUV-GUV interface where the protein initially accumulated.


( D ) Confocal microscopy images of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP . The arrows point to the concentrations of Atg5 - Atg12 / Atg16 on the GUV membrane . Scale bars : 10 μm ( A - C ) and 3 μm ( D ) .

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(D) Confocal microscopy images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the concentrations of Atg5-Atg12/Atg16 on the GUV membrane. Scale bars: 10 μm (A-C) and 3 μm (D).


( A ) Widefield images of rhodamine ‐ PE labelled GUVs 30 min GUVs cluster only in the presence of the Atg5 - Atg12 / Atg16 and Atg5 / Atg16 complexes .

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(A) Widefield images of rhodamine‐PE labelled GUVs 30 min GUVs cluster only in the presence of the Atg5-Atg12/Atg16 and Atg5/Atg16 complexes.


( B ) SUV aggregation assay using Folch liposomes and the indicated proteins . The proteins were added after a short delay . The symbols above , below the images or next to the curves represent Atg5 ( blue oval ) , Atg12 ( red circle ) and Atg16 ( green Y ) . The overall shape of the Atg5 - Atg12 / Atg16 complex is according to the structural model of the complex as proposed in Fujioka et al ( 2010 ) . Scale bars : 10 μm .

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(B) SUV aggregation assay using Folch liposomes and the indicated proteins. The proteins were added after a short delay. The symbols above, below the images or next to the curves represent Atg5 (blue oval), Atg12 (red circle) and Atg16 (green Y). The overall shape of the Atg5-Atg12/Atg16 complex is according to the structural model of the complex as proposed in Fujioka et al (2010). Scale bars: 10 μm.


( A ) Liposome co ‐ sedimentation assay using Folch liposomes and the indicated Atg5 proteins stabilized with an N ‐ terminal fragment of Atg16 ( amino acids 1 - 46 ) .

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(A) Liposome co‐sedimentation assay using Folch liposomes and the indicated Atg5 proteins stabilized with an N‐terminal fragment of Atg16 (amino acids 1-46).


( B ) Quantification based on three co ‐ sedimentation assays as shown in ( A ) . The amount of protein pelleted in the absence of liposomes was subtracted . The averages and the standard deviations are shown .

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(B) Quantification based on three co‐sedimentation assays as shown in (A). The amount of protein pelleted in the absence of liposomes was subtracted. The averages and the standard deviations are shown.


( C ) Yeast cells were transformed with the indicated expression constructs and Atg5 - 2 × Myc was immunoprecipitated with an anti ‐ Myc antibody . The precipitated protein was subjected to anti ‐ Atg5 and anti ‐ Atg12western blotting . The substitution of R171 and K160 to glutamate caused the mutant protein to run higher . The same phenomenon was observed for the recombinant protein ( Supplementary Figure 3 ) .

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(C) Yeast cells were transformed with the indicated expression constructs and Atg5-2 × Myc was immunoprecipitated with an anti‐Myc antibody. The precipitated protein was subjected to anti‐Atg5 and anti‐Atg12western blotting. The substitution of R171 and K160 to glutamate caused the mutant protein to run higher. The same phenomenon was observed for the recombinant protein ( Supplementary Figure 3).


( D ) Anti ‐ Atg12 western blot of cell fractions prepared from rapamycin ‐ treated yeast cells . The anti ‐ Pgk1 and anti ‐ Pex30 served as control for the cytosolic and membrane fractions , respectively .

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(D) Anti‐Atg12 western blot of cell fractions prepared from rapamycin‐treated yeast cells. The anti‐Pgk1 and anti‐Pex30 served as control for the cytosolic and membrane fractions, respectively.


( E ) Anti ‐ Myc and anti ‐ Pex30western blots of yeast cell fractions from cells of the indicated genotype and expressing Atg16 and Atg5 or Atg5 K160E , R171E .

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(E) Anti‐Myc and anti‐Pex30western blots of yeast cell fractions from cells of the indicated genotype and expressing Atg16 and Atg5 or Atg5 K160E, R171E.


( F ) Starvation assay with the indicated yeast strains and the indicated rescue constructs . GFP was detected by western blotting . Pgk1 served as loading control .

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(F) Starvation assay with the indicated yeast strains and the indicated rescue constructs. GFP was detected by western blotting. Pgk1 served as loading control.


( G ) Same assay as shown in ( F ) but using an anti ‐ Ape1 antiserum for western blotting .

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(G) Same assay as shown in (F) but using an anti‐Ape1 antiserum for western blotting.


( H ) Western blots of yeast cell lysates using an anti ‐ Atg8 antiserum .

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(H) Western blots of yeast cell lysates using an anti‐Atg8 antiserum.


( I ) Ape1 protease protection assay with the indicated yeast strains . The percentages below the blots indicate the percent of protected ApeI in the + protease K , − Triton X ‐ 100 lanes and are the average of three independent experiments ± the standard deviation . The numbers next to the blots indicate the molecular weight in kDa . Figure source data can be found with the Supplementary data .

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(I) Ape1 protease protection assay with the indicated yeast strains. The percentages below the blots indicate the percent of protected ApeI in the +protease K, −Triton X‐100 lanes and are the average of three independent experiments±the standard deviation. The numbers next to the blots indicate the molecular weight in kDa. Figure source data can be found with the Supplementary data.


( A ) Yeast cells of the indicated genotype expressing Atg5 - 3 × mCherry or Atg5 K160E , R171E - 3 × mCherry were treated with rapamycin and imaged using a Deltavision microscope . Maximum projections of z ‐ stacks are shown .

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(A) Yeast cells of the indicated genotype expressing Atg5-3 × mCherry or Atg5 K160E, R171E-3 × mCherry were treated with rapamycin and imaged using a Deltavision microscope. Maximum projections of z‐stacks are shown.


( B ) Graph showing the percentage of mCherry ‐ positive cells that display Atg5 - 3 × mCherry dots . In total , 3 independent experiments were conducted and > 400 cells were counted .

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(B) Graph showing the percentage of mCherry‐positive cells that display Atg5-3 × mCherry dots. In total, 3 independent experiments were conducted and >400 cells were counted.


( C ) Graph showing the percentage of cells that display Atg16 - 3 × mCherry dots . In total , 3 independent experiments were conducted and > 750 cells were counted . The graphs show the averages and the error bars representing the standard deviations . P ‐ values were calculated using Student ' s t ‐ test . Scale bars : 5 μm .

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(C) Graph showing the percentage of cells that display Atg16-3 × mCherry dots. In total, 3 independent experiments were conducted and >750 cells were counted. The graphs show the averages and the error bars representing the standard deviations. P‐values were calculated using Student's t‐test. Scale bars: 5 μm.


A A549 and U937 cells were infected with each IAV at MOI 1 or 5 for 24 h , and lysates were analyzed by Western blotting using polyclonal anti - PB1 - F2 antibody .

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A A549 and U937 cells were infected with each IAV at MOI 1 or 5 for 24 h, and lysates were analyzed by Western blotting using polyclonal anti-PB1-F2 antibody.


B A549 cells were transfected with a plasmid encoding Flag - tagged PB1 - F2 derived from the PR8 or 1918 strain and treated with the proteasome inhibitor MG132 ( 25 μM ) or vehicle for 6 h before harvesting . PB1 - F2 transcripts were analyzed by semi - quantitative RT - PCR ( top panel ) , and The PB1 - F2 protein was analyzed by Western blotting with monoclonal anti - Flag antibody .

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B A549 cells were transfected with a plasmid encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and treated with the proteasome inhibitor MG132 (25 μM) or vehicle for 6 h before harvesting. PB1-F2 transcripts were analyzed by semi-quantitative RT-PCR (top panel), and The PB1-F2 protein was analyzed by Western blotting with monoclonal anti-Flag antibody.


C Representative immunofluorescence images showing expression of PB1 - F2 ( red ) . A549 cells were transfected with plasmids encoding indicated PB1 - F2 . At 18 h after transfection , cells were treated with MG132 for 6 h and subjected to immunofluorescence analysis . Magnification , × 100 ; scale bar , 100 μm .

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C Representative immunofluorescence images showing expression of PB1-F2 (red). A549 cells were transfected with plasmids encoding indicated PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to immunofluorescence analysis. Magnification, ×100; scale bar, 100 μm.


D Each HA - tagged PB1 - F2 was transfected into 293T cells with or without Flag - ubiquitin . At 18 h after transfection , cells were treated with MG132 for 6 h . The lysates were immunoprecipitated with anti - HA antibody and analyzed by Western blotting with anti - Flag antibody . PR8 , A / Puerto Rico / 8 / 34 ( H1N1 ) ; 1918 , A / Brevig Mission / 1 / 1918 ( H1N1 ) .

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D Each HA-tagged PB1-F2 was transfected into 293T cells with or without Flag-ubiquitin. At 18 h after transfection, cells were treated with MG132 for 6 h. The lysates were immunoprecipitated with anti-HA antibody and analyzed by Western blotting with anti-Flag antibody. PR8, A/Puerto Rico/8/34 (H1N1); 1918, A/Brevig Mission/1/1918 (H1N1).


B - D A549 cells were transfected with the indicated plasmids encoding Flag - tagged PB1 - F2 derived from the PR8 or 1918 strain and chimeric PB1 - F2 mutants . At 18 h after transfection , cells were treated with or without MG132 for 6 h . The expression of PB1 - F2 and control genes was detected by semi - quantitative RT - PCR ( upper panel ) or Western blotting ( lower panel ) . Results shown are representative of three independent experiments .

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B-D A549 cells were transfected with the indicated plasmids encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and chimeric PB1-F2 mutants. At 18 h after transfection, cells were treated with or without MG132 for 6 h. The expression of PB1-F2 and control genes was detected by semi-quantitative RT-PCR (upper panel) or Western blotting (lower panel). Results shown are representative of three independent experiments.


E Representative immunofluorescence images of PB1 - F2 expression in A549 cells . Cells transfected with the Flag - tagged PB1 - F2 plasmid were stained with anti - Flag ( red ) antibody and analyzed by immunofluorescence assay . Magnification , × 400 ; scale bar , 50 μm .

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E Representative immunofluorescence images of PB1-F2 expression in A549 cells. Cells transfected with the Flag-tagged PB1-F2 plasmid were stained with anti-Flag (red) antibody and analyzed by immunofluorescence assay. Magnification, ×400; scale bar, 50 μm.


A Relative activity of NF - kB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1 - F2 . The mRNA levels of pro - inflammatory cytokines were determined by semi - quantitative RT - PCR . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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A Relative activity of NF-kB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1-F2. The mRNA levels of pro-inflammatory cytokines were determined by semi-quantitative RT-PCR. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


Cells were transfected with PB1 - F2 - expressing plasmids . Relative IFNβ mRNA levels were determined by semi - quantitative RT - PCR ( top ) and qPCR ( bottom ) . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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Cells were transfected with PB1-F2-expressing plasmids. Relative IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (bottom). All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


C U937 cells were co - transfected with the IFNβ luciferase reporter plasmid and the PB1 - F2 or NS1 expressing plasmid and treated with polyI : C for 12 h before harvesting . IFNβ promoter activity was determined by luciferase reporter assay . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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C U937 cells were co-transfected with the IFNβ luciferase reporter plasmid and the PB1-F2 or NS1 expressing plasmid and treated with polyI:C for 12 h before harvesting. IFNβ promoter activity was determined by luciferase reporter assay. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


Cells were infected for 24 h with each virus at 1 MOI . The mRNA level of IFNβ was determined by semi - quantitative RT - PCR , and PB1 - F2 protein expression was assessed by Western blotting . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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Cells were infected for 24 h with each virus at 1 MOI. The mRNA level of IFNβ was determined by semi-quantitative RT-PCR, and PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


E Cells were transfected with each plasmid encoding PB1 - F2 . 18 h after transfection , cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi - quantitative RT - PCR ( left ) and qPCR ( right ) . PB1 - F2 protein expression was assessed by Western blotting . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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E Cells were transfected with each plasmid encoding PB1-F2. 18 h after transfection, cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi-quantitative RT-PCR (left) and qPCR (right). PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


F A549 cells were transfected with PB1 - F2 clones mutated at positions 68 and 69 ; 24 h after transfection , the IFNβ mRNA level and PB1 - F2 protein expression were determined by semi - quantitative RT - PCR and Western blotting , respectively ( left ) . The relative expression level of IFNβ mRNA was also analyzed by qPCR ( right ) . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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F A549 cells were transfected with PB1-F2 clones mutated at positions 68 and 69; 24 h after transfection, the IFNβ mRNA level and PB1-F2 protein expression were determined by semi-quantitative RT-PCR and Western blotting, respectively (left). The relative expression level of IFNβ mRNA was also analyzed by qPCR (right). All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


A Mice ( n = 5 or 6 per group ) were infected with IAV with indicated pfu . Survival was monitored daily for 2 weeks .

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A Mice (n = 5 or 6 per group) were infected with IAV with indicated pfu. Survival was monitored daily for 2 weeks.


B Mice ( n = 6 - 9 per group ) were infected with each IAV as indicated . Body weight was recorded daily after infection for 2 weeks .

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B Mice (n = 6-9 per group) were infected with each IAV as indicated. Body weight was recorded daily after infection for 2 weeks.


C Mice ( n = 5 or 6 per group ) were infected with viruses ( 1000 pfu ) carrying PR8 PB1 - F2 with the I68T , L69P , or I68T + L69P mutation . Body weight changes and survival rate were monitored daily . Detailed analyses on the virulence necessitated the monitoring of body weight below the standard level ( 80 % ) of mortality .

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C Mice (n = 5 or 6 per group) were infected with viruses (1000 pfu) carrying PR8 PB1-F2 with the I68T, L69P, or I68T+L69P mutation. Body weight changes and survival rate were monitored daily. Detailed analyses on the virulence necessitated the monitoring of body weight below the standard level (80%) of mortality.


Three cohorts of mice ( 5 or 6 per group ) were infected with 500 pfu of one of the three IAVs . Viral protein expression in mouse lung was determined by Western blotting 2 days after infection .

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Three cohorts of mice (5 or 6 per group) were infected with 500 pfu of one of the three IAVs. Viral protein expression in mouse lung was determined by Western blotting 2 days after infection.


E Mice ( 6 per group ) were infected with 500 pfu of one of the three IAVs . Viral titration by plaque assay was performed at 2 days post - infection using lung homogenates .

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E Mice (6 per group) were infected with 500 pfu of one of the three IAVs. Viral titration by plaque assay was performed at 2 days post-infection using lung homogenates.


F Mice ( 5 - 9 per group ) were infected with 500 pfu of each virus . At 2 days post - infection , IFNβ mRNA levels were determined by qPCR .

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F Mice (5-9 per group) were infected with 500 pfu of each virus. At 2 days post-infection, IFNβ mRNA levels were determined by qPCR.


G Mice ( 5 - 9 per group ) were infected with 1 × 105 pfu of each virus . At 2 days post - infection , IFNβ protein levels in lung homogenates were measured by ELISA .

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G Mice (5-9 per group) were infected with 1 × 105 pfu of each virus. At 2 days post-infection, IFNβ protein levels in lung homogenates were measured by ELISA.


A SDS - PAGE analysis of proteins immunoprecipitated from A549 cell lysates using HA - antibody . Bands were stained by Coomassie Blue . Results were confirmed in three independent experiments , and representative data are shown .

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A SDS-PAGE analysis of proteins immunoprecipitated from A549 cell lysates using HA-antibody. Bands were stained by Coomassie Blue. Results were confirmed in three independent experiments, and representative data are shown.


E Analysis of interaction between DDX3 and 1918 PB1 - F2 . Cells were transfected with indicated plasmids ; 18 h later , cells were treated with MG132 for 6 h . Cell lysates were immunoprecipitated with anti - HA antibody . Proteins were detected by immunoblotting with the indicated antibodies ( left panel ) . IAV - infected cell lysates were immunoprecipitated with anti - PB1 - F2 antibody and immunoblotted with the indicated antibodies ( middle panel ) . Purified recombinant 1918 PB1 - F2 protein was incubated with recombinant LysRS or LysRS - DDX3 , and the interaction was analyzed by immunoblotting ( right panel ) . Results were confirmed in three independent experiments , and representative data are shown .

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E Analysis of interaction between DDX3 and 1918 PB1-F2. Cells were transfected with indicated plasmids; 18 h later, cells were treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-HA antibody. Proteins were detected by immunoblotting with the indicated antibodies (left panel). IAV-infected cell lysates were immunoprecipitated with anti-PB1-F2 antibody and immunoblotted with the indicated antibodies (middle panel). Purified recombinant 1918 PB1-F2 protein was incubated with recombinant LysRS or LysRS-DDX3, and the interaction was analyzed by immunoblotting (right panel). Results were confirmed in three independent experiments, and representative data are shown.


F Analysis of interaction between DDX3 and PB1 - F2 mutants . A549 cells were transfected with the indicated clones ; 18 h after transfection , cells were treated with MG132 for 6 h and analyzed by immunoprecipitation . Results were confirmed in three independent experiments , and representative data are shown .

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F Analysis of interaction between DDX3 and PB1-F2 mutants. A549 cells were transfected with the indicated clones; 18 h after transfection, cells were treated with MG132 for 6 h and analyzed by immunoprecipitation. Results were confirmed in three independent experiments, and representative data are shown.


G A549 cells were treated with IFNβ for the indicated time periods and subjected to Western blotting ( top ) . A549 cells were infected with indicated IAV . At 24 h post - infection , cell lysates were analyzed by Western blotting ( bottom ) . Results were confirmed in three independent experiments , and representative data are shown .

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G A549 cells were treated with IFNβ for the indicated time periods and subjected to Western blotting (top). A549 cells were infected with indicated IAV. At 24 h post-infection, cell lysates were analyzed by Western blotting (bottom). Results were confirmed in three independent experiments, and representative data are shown.


H Representative fluorescence images showing expression level of GFP - DDX3 ( green ) . A549 cells were co - transfected with plasmids encoding the GEP - DDX3 and Flag - tagged PR8 or 1918 PB1 - F2 . At 18 h after transfection , cells were treated with MG132 for 6 h and subjected to fluorescence analysis . Results were confirmed in three independent experiments , and representative data are shown .

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H Representative fluorescence images showing expression level of GFP-DDX3 (green). A549 cells were co-transfected with plasmids encoding the GEP-DDX3 and Flag-tagged PR8 or 1918 PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to fluorescence analysis. Results were confirmed in three independent experiments, and representative data are shown.


I Mice were infected with 1 × 105 pfu of each virus , and lung homogenates were examined for endogenous Ddx3 expression . Results were confirmed in three independent experiments , and representative data are shown .

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I Mice were infected with 1×105 pfu of each virus, and lung homogenates were examined for endogenous Ddx3 expression. Results were confirmed in three independent experiments, and representative data are shown.


J A549 cells were transfected with the indicated plasmids or treated with poly I : C . Cytoplasmic and nuclear fractions were separated and subjected to Western blotting . Results were confirmed in three independent experiments , and representative data are shown .

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J A549 cells were transfected with the indicated plasmids or treated with poly I:C. Cytoplasmic and nuclear fractions were separated and subjected to Western blotting. Results were confirmed in three independent experiments, and representative data are shown.


K A549 cells were transfected with the indicated plasmids , and IFNβ mRNA levels were determined by semi - quantitative RT - PCR ( top ) and qPCR ( middle ) . IFNβ promoter activity was determined by luciferase reporter assay ( bottom ) . Results were confirmed in three independent experiments , and representative data are shown .

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K A549 cells were transfected with the indicated plasmids, and IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (middle). IFNβ promoter activity was determined by luciferase reporter assay (bottom). Results were confirmed in three independent experiments, and representative data are shown.


Groups of five mice were intranasally infected with IAV ( 1918 ) , and recombinant DDX3 protein was administered intranasally . Survival was monitored daily for 2 weeks .

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Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Survival was monitored daily for 2 weeks.


Groups of five mice were intranasally infected with IAV ( 1918 ) , and recombinant DDX3 protein was administered intranasally . Two days post - infection , virus titers in lung homogenates were determined by plaque assay . A p value of < 0 . 05 was considered significant ( * * p < 0 . 01 ) .

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Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Two days post-infection, virus titers in lung homogenates were determined by plaque assay. A p value of <0.05 was considered significant (** p < 0.01).


Groups of five mice were intranasally infected with IAV ( 1918 ) , and recombinant DDX3 protein was administered intranasally . Two days post - infection , the expression level of IFNβ in the lungs was analyzed by semi - quantitative RT - PCR ( left panel ) and qPCR ( right panel ) . A p value of < 0 . 05 was considered significant ( * * p < 0 . 01 ) .

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Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Two days post-infection, the expression level of IFNβ in the lungs was analyzed by semi-quantitative RT-PCR (left panel) and qPCR (right panel). A p value of <0.05 was considered significant (** p < 0.01).


( B ) Left , orthogonal view of a 20μm tumor section labelled with antibodies against EpCAM ( in red ) and DNA in blue . Right , dot plot bar representing cell heights in cells from tumor sections , n = 60 cells from 1 tumor .

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(B) Left, orthogonal view of a 20μm tumor section labelled with antibodies against EpCAM (in red) and DNA in blue. Right, dot plot bar representing cell heights in cells from tumor sections, n=60 cells from 1 tumor.


( C ) On the left , images of low magnification views of healthy tissues labelled with antibodies against pericentrin ( PCNT ) and CDK5RAP2 , shown in red and green , respectively . DNA is in blue . The white dashed squares represent the regions shown in higher magnifications on the right . One centrosome was considered as such when PCNT and CDK5RAP2 signals co - localized ( Inset 1 ) . Lack of co - localization ( Insets 2 and 3 ) was noticed and discarded during quantification .

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(C) On the left, images of low magnification views of healthy tissues labelled with antibodies against pericentrin (PCNT) and CDK5RAP2, shown in red and green, respectively. DNA is in blue. The white dashed squares represent the regions shown in higher magnifications on the right. One centrosome was considered as such when PCNT and CDK5RAP2 signals co-localized (Inset 1). Lack of co-localization (Insets 2 and 3) was noticed and discarded during quantification.


( D ) Super - resolution microscopy of healthy tissues labelled for the centriole marker CEP135 ( in green ) and PCNT ( in red ) .

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(D) Super-resolution microscopy of healthy tissues labelled for the centriole marker CEP135 (in green) and PCNT (in red).


( E - F ) Representative images of two different fields of the same tumor ( E ) and of a tumor section to highlight different centrososome phenotypes , labelled as described in ( C ) . The pink dashed line highlights an area of cells without centrosomes . The white dashed squares represent the regions shown in higher magnifications in insets in ( G and I ) and the pink dashed linesurrounds nuclei without centrosomes . ( G ) Insets from ( F ) . ( H ) Examples of tumor sections showing small ( left ) and large ( right ) regions with nuclei without centrosomes .

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(E-F) Representative images of two different fields of the same tumor (E) and of a tumor section to highlight different centrososome phenotypes, labelled as described in (C). The pink dashed line highlights an area of cells without centrosomes. The white dashed squares represent the regions shown in higher magnifications in insets in (G and I) and the pink dashed linesurrounds nuclei without centrosomes. (G) Insets from (F). (H) Examples of tumor sections showing small (left) and large (right) regions with nuclei without centrosomes.


( I ) Insets from ( F ) showing different types of centrosome amplification events .

(I) Insets from (F) showing different types of centrosome amplification events.


( J ) Super - resolution microscopy of tumor tissues labelled as in ( C ) .

(J) Super-resolution microscopy of tumor tissues labelled as in (C).


( K ) Maximum Z - projection of a tumor section labelled with EpCAM antibodies ( Red ) and DNA in blue . The dashed line shows the absence of EpCAM staining , arrows show a disorganized region .

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(K) Maximum Z-projection of a tumor section labelled with EpCAM antibodies (Red) and DNA in blue. The dashed line shows the absence of EpCAM staining, arrows show a disorganized region.


( A ) Images of EOC tumors labelled with antibodies against PCNT ( red ) and CDKRAP2 ( green ) . Nuclei are shown in blue . The colored arrows reflect different centrosome number . The list of centrosome behaviors annotated it is not exhaustive to allow comprehension of the events . # TT72 , shows a cyst - like arrangement of EOC nuclei where it is impossible to correspond centrosomes to individual nuclei . The white bars with arrows of different colors highlight possible interpretations of the same condition . In # TT79 , regions without centrosomes can be identified . In the inset , a single centrosome localized in the lumen can be identified and it is not possible to know to which nucleus ( white dashed lines ) it corresponds to . # TT88 presents only very small clusters of centrosomes , few nuclei without centrosomes and many conditions with 2 centrosomes . In # TT61 , regions without centrosomes can be distinguished , in addition to nuclei associated with single centrosomes . In # TT46 , clusters containing large ( inset 1 ) or small clusters ( inset 3 ) can be identified . In cyst - like arrangements several centrosomes can be identified ( inset 2 ) , suggesting that all cells have at least one centrosome or , in contrast just one centrosome ( inset 4 ) , illustrating lack of centrosomes .

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(A) Images of EOC tumors labelled with antibodies against PCNT (red) and CDKRAP2 (green). Nuclei are shown in blue. The colored arrows reflect different centrosome number. The list of centrosome behaviors annotated it is not exhaustive to allow comprehension of the events. #TT72, shows a cyst-like arrangement of EOC nuclei where it is impossible to correspond centrosomes to individual nuclei. The white bars with arrows of different colors highlight possible interpretations of the same condition. In #TT79, regions without centrosomes can be identified. In the inset, a single centrosome localized in the lumen can be identified and it is not possible to know to which nucleus (white dashed lines) it corresponds to. #TT88 presents only very small clusters of centrosomes, few nuclei without centrosomes and many conditions with 2 centrosomes. In #TT61, regions without centrosomes can be distinguished, in addition to nuclei associated with single centrosomes. In #TT46, clusters containing large (inset 1) or small clusters (inset 3) can be identified. In cyst-like arrangements several centrosomes can be identified (inset 2), suggesting that all cells have at least one centrosome or, in contrast just one centrosome (inset 4), illustrating lack of centrosomes.