( E ) IF analyses of key markers as described in Fig 1E in the cultured cy rOvaries . Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper - right magnified images of each panel show the expression of key markers ( green ) co - stained with DDX4 ( magenta ) . The lower - right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) . The numbers written in the upper - left corner indicate the period of IVC ( w - ivc ) . Scale bar = 20 μm . ( F ) Percentages of cells positive for individual markers among DDX4 + germ cells from the IF of cy rOvaries at 3 - 15 w - ivc [ 3 / 6 / 9 / 12 w - ivc ( n = 3 ) , 15 w - ivc ( n = 3 or 4 ) ] .

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

(E) IF analyses of key markers as described in Fig 1E in the cultured cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate the period of IVC (w-ivc). Scale bar = 20 μm. (F) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3-15 w-ivc [3/6/9/12 w-ivc (n=3), 15 w-ivc (n=3 or 4)].


( A ) Gross appearance ( a ) , H & E staining ( b ) , and IF ( c ) for FOXL2 / DDX4 / DAPI of the human in vivo female gonads ( 11W , left column ) and the cultured rOvaries at 7 and 14 w - ivc . FOXL2 , granulosa cell marker ; DDX4 , germ cell marker ; DAPI , nucleus . Scale bars = 1 mm ( a , left ) , 200 μm ( a , middle / right ) , 50 μm ( b ) , and 20 μm ( c ) . g . a . , gestational age .

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

(A) Gross appearance (a), H&E staining (b), and IF (c) for FOXL2/DDX4/DAPI of the human in vivo female gonads (11W, left column) and the cultured rOvaries at 7 and 14 w-ivc. FOXL2, granulosa cell marker; DDX4, germ cell marker; DAPI, nucleus. Scale bars = 1 mm (a, left), 200 μm (a, middle/right), 50 μm (b), and 20 μm (c). g.a., gestational age.


IF for oogonia ( POU5F1 ) , cell proliferation ( Ki67 ) , meiotic ( SYCP3 , DMC1 , γH2AX , SYCP1 ) , and oogenic ( ZP3 , TP63 ) markers for human in vivo female gonad ( 11W , B ) Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper right magnified images of each panel show the expression of key markers ( green ) co - stained with DDX4 ( magenta ) . The lower right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) .

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

IF for oogonia (POU5F1), cell proliferation (Ki67), meiotic (SYCP3, DMC1, γH2AX, SYCP1), and oogenic (ZP3, TP63) markers for human in vivo female gonad (11W, B) Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white).


IF for oogonia ( POU5F1 ) , cell proliferation ( Ki67 ) , meiotic ( SYCP3 , DMC1 , γH2AX , SYCP1 ) , and oogenic ( ZP3 , TP63 ) markers human in vitro cultured rOvaries at 14 w - ivc ( C ) . Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper right magnified images of each panel show the expression of key markers ( green ) co - stained with DDX4 ( magenta ) . The lower right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) .

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

IF for oogonia (POU5F1), cell proliferation (Ki67), meiotic (SYCP3, DMC1, γH2AX, SYCP1), and oogenic (ZP3, TP63) markers human in vitro cultured rOvaries at 14 w-ivc (C). Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white).


( D ) Percentages of cells positive for individual markers among DDX4 + germ cells from the IF of human in vivo female gonads ( 11W ) and human cultured rOvaries at 7 and 14 w - ivc . The mean values from two biological replicates are shown .

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 O O O

(D) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of human in vivo female gonads (11W) and human cultured rOvaries at 7 and 14 w-ivc. The mean values from two biological replicates are shown.


Analyses for human germ cells by SC3 - seq . ( E ) UHC for human germ cells in vivo and in rOvaries with all expressed genes ( 20 , 048 genes ) and a heatmap of the expression levels of selected marker genes . Color bars under the dendrogram show the in vivo stage ( 1st bar ) , the period of IVC ( 2nd bar ) , and the cell type ( 3rd bar ) . The heatmap color - coding is as indicated . The color coding in bars is as indicated in Fig 5F .

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

Analyses for human germ cells by SC3-seq. (E) UHC for human germ cells in vivo and in rOvaries with all expressed genes (20,048 genes) and a heatmap of the expression levels of selected marker genes. Color bars under the dendrogram show the in vivo stage (1st bar), the period of IVC (2nd bar), and the cell type (3rd bar). The heatmap color-coding is as indicated. The color coding in bars is as indicated in Fig 5F.


Analyses performed by 10X scRNA - seq . ( A ) UMAP plot of germ cells from six 10X scRNA - seq datasets ( two for humans [ this study and ( Chitiashvili et al . , 2020 ) ] , one for monkeys [ this study ] , and three for mice [ ( Ge et al . , 2021 , Niu & Spradling , 2020 , Zhao et al . , 2020 ) ] ) integrated by CCA . Meiotic sub - stages were defined after CCA followed by Louvain clustering . Colors indicate the computationally assigned meiotic sub - stages defined by the expression levels of key meiotic markers conserved in all three species ( see Materials and Methods ) . The color - coding is as indicated . M , mitotic ; PL , pre - leptotene ; L , leptotene ; Z , zygotene ; P , pachytene ; D , diplotene . ( B ) The UMAP plot highlighting cells from each dataset . The color - coding for each developmental stage is as 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analyses performed by 10X scRNA-seq. (A) UMAP plot of germ cells from six 10X scRNA-seq datasets (two for humans [this study and (Chitiashvili et al., 2020)], one for monkeys [this study], and three for mice [(Ge et al., 2021, Niu & Spradling, 2020, Zhao et al., 2020)]) integrated by CCA. Meiotic sub-stages were defined after CCA followed by Louvain clustering. Colors indicate the computationally assigned meiotic sub-stages defined by the expression levels of key meiotic markers conserved in all three species (see Materials and Methods). The color-coding is as indicated. M, mitotic; PL, pre-leptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene. (B) The UMAP plot highlighting cells from each dataset. The color-coding for each developmental stage is as indicated.


( I ) IF analyses of CLGN with TP63 ( oogenic marker ) and DDX4 ( germ cell marker ) in a 13 - year - old human oocyte of a primordial follicle . Nuclear DAPI staining is shown in white .

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

(I) IF analyses of CLGN with TP63 (oogenic marker) and DDX4 (germ cell marker) in a 13-year-old human oocyte of a primordial follicle. Nuclear DAPI staining is shown in white.


( C ) The Chr . 10 : A and X : A ratios ( top ) , and XIST expression ( bottom ) in human female germ cells in vivo and in cultured rOvaries analyzed by SC3 - seq .

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

(C) The Chr.10:A and X:A ratios (top), and XIST expression (bottom) in human female germ cells in vivo and in cultured rOvaries analyzed by SC3-seq.


A HeLa cells expressing LAMP1 - RFP were treated with vehicle alone ( control ) or LLOMe for 1 h to induce lysosome damage . Cells were fixed 2 h after washout , stained with p62 and galectin - 3 ( Gal3 ) antibodies , and analyzed by confocal microscopy .

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

A HeLa cells expressing LAMP1-RFP were treated with vehicle alone (control) or LLOMe for 1 h to induce lysosome damage. Cells were fixed 2 h after washout, stained with p62 and galectin-3 (Gal3) antibodies, and analyzed by confocal microscopy.


B p97 translocates to ruptured lysosomes . Cells co - expressing LAMP1 - RFP and GFP - p97 were treated as in ( A ) and stained with the pan - ubiquitin antibody FK2 ( Ub ) . Percentage of cells showing recruitment of p97 in three independent experiments ( mean ± SD ) .

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

B p97 translocates to ruptured lysosomes. Cells co-expressing LAMP1-RFP and GFP-p97 were treated as in (A) and stained with the pan-ubiquitin antibody FK2 (Ub). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).


C Immunodetection of endogenous p97 after LLOMe treatment as in ( A ) . Percentage of cells showing recruitment of p97 in three independent experiments ( mean ± SD ) .

O O O O B-MEASURED_VAR O B-CONTROLLED_VAR 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

C Immunodetection of endogenous p97 after LLOMe treatment as in (A). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).


D Clearance of damaged lysosomes depends on p97 . Cells were transfected with control ( Ctrl ) or p97 siRNA oligos , fixed at indicated time points after LLOMe washout and stained for endogenous Gal3 . Quantification of the number of Gal3 - positive vesicles per cell . Data represent mean ± SD of four independent experiments with ≥ 30 cells quantified per condition ( one - sided Welch ' s unequal variances t - tests comparing the siCtrl with sip97 ) .

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

D Clearance of damaged lysosomes depends on p97. Cells were transfected with control (Ctrl) or p97 siRNA oligos, fixed at indicated time points after LLOMe washout and stained for endogenous Gal3. Quantification of the number of Gal3-positive vesicles per cell. Data represent mean ± SD of four independent experiments with ≥30 cells quantified per condition (one-sided Welch's unequal variances t-tests comparing the siCtrl with sip97).


E Cells were chased after LLOMe treatment for 12 h with vehicle alone ( DMSO ) , bortezomib ( Btz , 10 nM ) , NH4Cl ( 20 mM ) or the p97 inhibitor NMS - 873 ( 5 µM ) , and Gal3 vesicles quantified . Data represent mean ± SEM of three independent experiments with ≥ 30 cells quantified per condition ( one - sided Welch ' s t - tests ) .

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

E Cells were chased after LLOMe treatment for 12 h with vehicle alone (DMSO), bortezomib (Btz, 10 nM), NH4Cl (20 mM) or the p97 inhibitor NMS-873 (5 µM), and Gal3 vesicles quantified. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition (one-sided Welch's t-tests).


F p97 is essential for cell survival after lysosome rupture . Cells were transfected with control ( Ctrl ) , p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h . Cell viability was measured using the MTS assay . Data represent mean ± SD of three independent experiments .

O B-CONTROLLED_VAR 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 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

F p97 is essential for cell survival after lysosome rupture. Cells were transfected with control (Ctrl), p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h. Cell viability was measured using the MTS assay. Data represent mean ± SD of three independent experiments.


G Taufibrils are endocytosed and induce lysosomal damage . Taufibrils were generated in vitro , fluorescently labelled by Dylight488 and fragmented by sonication ( 488 - labelled tau ) . HeLa cells expressing mCherry - Gal3 were incubated with 300 nM taufibrils for 24 h . Arrows indicate compartments that contain 488 - labelled tau and are positive for mCherry - Gal3 indicating lysosome rupture . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD ) .

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

G Taufibrils are endocytosed and induce lysosomal damage. Taufibrils were generated in vitro, fluorescently labelled by Dylight488 and fragmented by sonication (488-labelled tau). HeLa cells expressing mCherry-Gal3 were incubated with 300 nM taufibrils for 24 h. Arrows indicate compartments that contain 488-labelled tau and are positive for mCherry-Gal3 indicating lysosome rupture. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD).


H Accumulation of tau - induced lysosomal damage upon p97 depletion . Cells transfected with control ( Ctrl ) or p97 siRNA oligos were treated as in ( G ) . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD ) . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 . Scale bars , 10 µm .

O O O B-MEASURED_VAR O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O 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 Accumulation of tau-induced lysosomal damage upon p97 depletion. Cells transfected with control (Ctrl) or p97 siRNA oligos were treated as in (G). Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD). * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Scale bars, 10 µm.


A Impaired clearance of damaged lysosomes in cells expressing a p97 disease - mutant . Wild - type and p97R155H / + MEFs were LLOMe - treated for 1 h and released for indicated times . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD , Student ' s unpaired t - test ) . Scale bar , 25 µm .

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

A Impaired clearance of damaged lysosomes in cells expressing a p97 disease-mutant. Wild-type and p97R155H/+MEFs were LLOMe-treated for 1 h and released for indicated times. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD, Student's unpaired t-test). Scale bar, 25 µm.


B Stable U2OS cell lines were doxycycline - induced to express myc - tagged p97 wild - type or disease - mutants as indicated . Induced parental cells served as control ( U2OS - ) . Cells were treated with LLOMe for 1h and chased for indicated time before fixation . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments . Comparable expression levels were verified ( see Figure EV2A ) .

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 B-CONTROLLED_VAR 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

B Stable U2OS cell lines were doxycycline-induced to express myc-tagged p97 wild-type or disease-mutants as indicated. Induced parental cells served as control (U2OS -). Cells were treated with LLOMe for 1h and chased for indicated time before fixation. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments. Comparable expression levels were verified (see Figure EV2A).


C Stable U2OS cell lines described in ( B ) were treated with LLOMe for 1 h and chased for indicated time . LC3 and GAPDH levels were analyzed by immunoblotting . Quantification is shown in Figure EV2B .

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

C Stable U2OS cell lines described in (B) were treated with LLOMe for 1 h and chased for indicated time. LC3 and GAPDH levels were analyzed by immunoblotting. Quantification is shown in Figure EV2B.


D Accumulation of damaged lysosomes in affected p97 patient skeletal muscle . Patient biopsies immunostained for Gal3 and caveolin - 3 ( Cav3 ) . Quantification from 4 random 10X fields for each biopsy and means for controls and patients , respectively . Patients carry p97 mutations R155H ( # 1 - 3 ) or R93C for ( # 4 ) . Patients # 2 and # 3 are cousins . Scale bar , 100 µm .

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

D Accumulation of damaged lysosomes in affected p97 patient skeletal muscle. Patient biopsies immunostained for Gal3 and caveolin-3 (Cav3). Quantification from 4 random 10X fields for each biopsy and means for controls and patients, respectively. Patients carry p97 mutations R155H (#1-3) or R93C for (#4). Patients #2 and #3 are cousins. Scale bar, 100 µm.


A Candidate screen for p97 cofactors involved in autophagy of damaged lysosomes . HeLa cells were transfected with indicated siRNA oligos and LLOMe - treated for 1 h . Cells were chased for 12 h , fixed and stained for Gal3 . See Figure EV3 for depletion efficiency . B Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition . This threshold was chosen so that less than 1 % of untreated cells were considered positive . Data represent mean ± SEM of three independent experiments with ≥ 30 cells quantified per condition . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - sided Welch ' s t - tests ) .

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-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 O O

A Candidate screen for p97 cofactors involved in autophagy of damaged lysosomes. HeLa cells were transfected with indicated siRNA oligos and LLOMe-treated for 1 h. Cells were chased for 12 h, fixed and stained for Gal3. See Figure EV3 for depletion efficiency.B Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition. This threshold was chosen so that less than 1% of untreated cells were considered positive. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-sided Welch's t-tests).


C YOD1 , UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes . Cells expressing indicated tagged proteins were LLOMe - treated for 1 h or with vehicle alone ( control ) , fixed 2 h after washout , and stained for endogenous LAMP1 and , in the case of PLAA , with Alexa - 488 conjugated anti - HA antibodies . See Figure EV3B for quantification .

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

C YOD1, UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes. Cells expressing indicated tagged proteins were LLOMe-treated for 1 h or with vehicle alone (control), fixed 2 h after washout, and stained for endogenous LAMP1 and, in the case of PLAA, with Alexa-488 conjugated anti-HA antibodies. See Figure EV3B for quantification.


D An ATP - stabilized complex of p97 containing both UBXD1 and PLAA . Stable HEK293 cell lines were doxycycline - induced to express p97 wild - type ( wt ) or the ATPase - mutant E578Q ( EQ ) at near endogenous level . Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies . Arrowheads indicate endogenous ( lower band ) and induced p97 ( upper band ) .

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

D An ATP-stabilized complex of p97 containing both UBXD1 and PLAA. Stable HEK293 cell lines were doxycycline-induced to express p97 wild-type (wt) or the ATPase-mutant E578Q (EQ) at near endogenous level. Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies. Arrowheads indicate endogenous (lower band) and induced p97 (upper band).


E Stable HEK293 cells treated as in ( D ) and transiently expressing GFP - tagged YOD1 - wt or the YOD1 - C160S catalytic mutant ( CS ) were processed for co - immunoprecipitation with GFP nanobodies . Asterisk , unspecific band . Note that the YOD1 - CS mutant stabilized p97 binding and , in the p97 - EQ background , also associated with UBXD1 and PLAA .

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

E Stable HEK293 cells treated as in (D) and transiently expressing GFP-tagged YOD1-wt or the YOD1-C160S catalytic mutant (CS) were processed for co-immunoprecipitation with GFP nanobodies. Asterisk, unspecific band. Note that the YOD1-CS mutant stabilized p97 binding and, in the p97-EQ background, also associated with UBXD1 and PLAA.


F Control ( IgG ) and UBXD1co - immunoprecipitation with indicated associated proteins from HeLa cells expressing GFP - YOD1 - CS after formaldehyde cross - linking . Ufd1 served as a negative control . Scale bars , 10 µm .

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

F Control (IgG) and UBXD1co-immunoprecipitation with indicated associated proteins from HeLa cells expressing GFP-YOD1-CS after formaldehyde cross-linking. Ufd1 served as a negative control. Scale bars, 10 µm.


A K63 - and K48 - ubiquitination of damaged lysosomes . Untransfected HeLa cells , or cells expressing mCherry - Gal3 ( Ch - Gal3 ) were LLOMe - treated for 1 h . 2 h after washout , cells were stained with antibodies specific for K63 or K48 chains , and LAMP1 or p62 as indicated . Percentage of K63 - or K48 - ubiquitinated LAMP1 vesicles and percentage of p62 - positive K63 or K48 vesicles were quantified from three independent experiments ( mean ± SD , Student ' s unpaired t - test for LAMP1 ) . * = p < 0 . 05 .

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

A K63- and K48-ubiquitination of damaged lysosomes. Untransfected HeLa cells, or cells expressing mCherry-Gal3 (Ch-Gal3) were LLOMe-treated for 1 h. 2 h after washout, cells were stained with antibodies specific for K63 or K48 chains, and LAMP1 or p62 as indicated. Percentage of K63- or K48-ubiquitinated LAMP1 vesicles and percentage of p62-positive K63 or K48 vesicles were quantified from three independent experiments (mean ± SD, Student's unpaired t-test for LAMP1). * = p < 0.05.


B Colocalization of K63 - and K48 - linked ubiquitin conjugates on individual lysosomes . Cells expressing GFP - TAB2 - NZF ( K63 - sensor ) were treated as in ( A ) and stained for K48 chains and LAMP1 . Note that the apparent nuclear localization of the sensor is a consequence of the extraction procedure ( van Wijk et al . , 2012 ) .

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

B Colocalization of K63- and K48-linked ubiquitin conjugates on individual lysosomes. Cells expressing GFP-TAB2-NZF (K63-sensor) were treated as in (A) and stained for K48 chains and LAMP1. Note that the apparent nuclear localization of the sensor is a consequence of the extraction procedure (van Wijk et al., 2012).


C Cells expressing GFP - p97 - EQ were treated as in ( A ) and stained with antibodies as indicated . D Quantification of the total number of K63 - or K48 - ubiquitinated vesicles per cell , and number of p97 - EQ positive K63 - or K48 - ubiquitinated vesicles ( mean ± SD ) . E Percentage of GFP - p97 - EQ vesicles positive for K63 or K48 chains was determined ( mean ± SD ) .

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

C Cells expressing GFP-p97-EQ were treated as in (A) and stained with antibodies as indicated.D Quantification of the total number of K63- or K48-ubiquitinated vesicles per cell, and number of p97-EQ positive K63- or K48-ubiquitinated vesicles (mean ± SD).E Percentage of GFP-p97-EQ vesicles positive for K63 or K48 chains was determined (mean ± SD).


F ELDR components target K48 - conjugates on lysosomes . Detection of GFP - YOD1 - CS or UBXD1 - GFP alone , or co - transfected with PLAA - HA and GFP - p97 - EQ , 2 h after LLOMe washout and co - stained with indicated antibodies .

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

F ELDR components target K48-conjugates on lysosomes. Detection of GFP-YOD1-CS or UBXD1-GFP alone, or co-transfected with PLAA-HA and GFP-p97-EQ, 2 h after LLOMe washout and co-stained with indicated antibodies.


A Time course of K63 - and K48 - ubiquitination . Control - depleted cells were fixed at indicated time points after LLOMe washout and stained with chain - specific antibodies and LAMP1 . Automated quantification of LAMP1 vesicles positive for K63 or K48 chains . Data represent mean ± SD of three independent experiments . B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes . Experiment and quantification as in ( A ) in cells treated with p97 or YOD1 siRNA oligos .

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

A Time course of K63- and K48-ubiquitination. Control-depleted cells were fixed at indicated time points after LLOMe washout and stained with chain-specific antibodies and LAMP1. Automated quantification of LAMP1 vesicles positive for K63 or K48 chains. Data represent mean ± SD of three independent experiments.B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes. Experiment and quantification as in (A) in cells treated with p97 or YOD1 siRNA oligos.


C The p97 - EQ mutant stabilizes K48 - conjugates . Lysosome damage was induced as in ( A ) in cells transfected with GFP - tagged p97 - wt or p97 - EQ . Cells were chased after washout for 12 h and stained for K48 chains . D Quantification of K48 - positive lysosomes 2 h and 12 h after damage in cells treated as in ( A ) . Data represent mean ± SD of three independent experiments .

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 B-MEASURED_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 O 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 The p97-EQ mutant stabilizes K48-conjugates. Lysosome damage was induced as in (A) in cells transfected with GFP-tagged p97-wt or p97-EQ. Cells were chased after washout for 12 h and stained for K48 chains.D Quantification of K48-positive lysosomes 2 h and 12 h after damage in cells treated as in (A). Data represent mean ± SD of three independent experiments.


E The catalytically - inactive mutant of YOD1 stabilizes K48 - conjugates . Cells transfected with GFP - tagged YOD1 - wt or the YOD1 - CS were treated and stained as in ( C ) . F Quantification as in ( D ) of cells treated as in ( E ) . * * = p < 0 . 01 * * * = p < 0 . 001 ( Student ' s unpaired t - test ) . ns , not significant . Scale bars , 10 µm .

O O O O O O O B-MEASURED_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

E The catalytically-inactive mutant of YOD1 stabilizes K48-conjugates. Cells transfected with GFP-tagged YOD1-wt or the YOD1-CS were treated and stained as in (C).F Quantification as in (D) of cells treated as in (E). ** = p < 0.01 *** = p < 0.001 (Student's unpaired t-test). ns, not significant. Scale bars, 10 µm.


A YOD1 can link p97 to ubiquitin . Ubiquitin - GST ( Ub - GST ) or GST alone was incubated with the indicated purified proteins and isolated . Co - isolated proteins ( pulldown ) were detected in SDS - gels . Note binding of p97 and YOD1 to Ub - GST only in combination .

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

A YOD1 can link p97 to ubiquitin. Ubiquitin-GST (Ub-GST) or GST alone was incubated with the indicated purified proteins and isolated. Co-isolated proteins (pulldown) were detected in SDS-gels. Note binding of p97 and YOD1 to Ub-GST only in combination.


B The S2 site of YOD1 is critical for ubiquitin binding . Experiments as in ( A ) with indicated combinations of p97 and YOD1 - wt or the ubiquitin - binding mutant , MutS2 .

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

B The S2 site of YOD1 is critical for ubiquitin binding. Experiments as in (A) with indicated combinations of p97 and YOD1-wt or the ubiquitin-binding mutant, MutS2.


C Reconstitution of a complex of ELDR components on ubiquitin . Experiments as in ( A ) with combinations of p97 - wt or p97 - EQ , YOD1 , PLAA and UBXD1 as indicated . Note that PLAA associates with p97 and YOD1 on ubiquitin only in the case of p97 - EQ .

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

C Reconstitution of a complex of ELDR components on ubiquitin. Experiments as in (A) with combinations of p97-wt or p97-EQ, YOD1, PLAA and UBXD1 as indicated. Note that PLAA associates with p97 and YOD1 on ubiquitin only in the case of p97-EQ.


D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site . LLOMe - treated cells expressing GFP - YOD1 - CS or the double mutant GFP - YOD1 - CS - MutS2 were chased for 2 h and stained for K48 chains and LAMP1 . Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined .

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

D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site. LLOMe-treated cells expressing GFP-YOD1-CS or the double mutant GFP-YOD1-CS-MutS2 were chased for 2 h and stained for K48 chains and LAMP1. Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined.


E The S2 site is essential for K48 - conjugate removal . GFP - YOD1 - wt or GFP - YOD1 - MutS2 overexpressing cells treated as in ( D ) were stained for K48 chains . Quantification of the number of K48 - positive vesicles per cell . Data represent mean ± SD of three independent experiments . * * = p < 0 . 01 ( Student ' s unpaired t - test ) . Scale bars , 10 µm .

O O O O O O O O O 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

E The S2 site is essential for K48-conjugate removal. GFP-YOD1-wt or GFP-YOD1-MutS2 overexpressing cells treated as in (D) were stained for K48 chains. Quantification of the number of K48-positive vesicles per cell. Data represent mean ± SD of three independent experiments. ** = p < 0.01 (Student's unpaired t-test). Scale bars, 10 µm.


A Largely mutually exclusive localization of LC3 and K48 chains . GFP - LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains . Correlation coefficients were calculated for colocalization of LC3 with K63 or K48 . The whiskers represent 10th and 90th percentile . Points below and above the whiskers are drawn as individual dots . The single line indicates the median . * * * = p < 0 . 001 ( Mann - Whitney - U test ) .

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

A Largely mutually exclusive localization of LC3 and K48 chains. GFP-LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains. Correlation coefficients were calculated for colocalization of LC3 with K63 or K48. The whiskers represent 10th and 90th percentile. Points below and above the whiskers are drawn as individual dots. The single line indicates the median. *** = p < 0.001 (Mann-Whitney-U test).


B Lipofectamine - coated latex beads damage lysosomes and induce their K48 - ubiquitination . Cells expressing mCherry - Gal3 were incubated with Lipofectamine - coated latex beads for 3 h and stained with K48 chain - specific antibodies .

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

B Lipofectamine-coated latex beads damage lysosomes and induce their K48-ubiquitination. Cells expressing mCherry-Gal3 were incubated with Lipofectamine-coated latex beads for 3 h and stained with K48 chain-specific antibodies.


C YOD1 association and dissociation precedes LC3 recruitment . Live cell video microscopy stills of cells co - expressing mCherry - YOD1 - wt and GFP - LC3 after treatment with Lipofectamine - coated latex beads for 30 min at 2 min image intervals .

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

C YOD1 association and dissociation precedes LC3 recruitment. Live cell video microscopy stills of cells co-expressing mCherry-YOD1-wt and GFP-LC3 after treatment with Lipofectamine-coated latex beads for 30 min at 2 min image intervals.


D Stabilization of K48 - conjugates on lysosomes impairs recruitment of LC3 . Cells expressing GFP - YOD1 - CS were treated as in ( A ) and stained for LAMP1 and p62 or LC3 . Percentage of GFP - YOD1 vesicles positive for p62 or LC3 was determined ( mean ± SD ) .

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

D Stabilization of K48-conjugates on lysosomes impairs recruitment of LC3. Cells expressing GFP-YOD1-CS were treated as in (A) and stained for LAMP1 and p62 or LC3. Percentage of GFP-YOD1 vesicles positive for p62 or LC3 was determined (mean ± SD).


E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation . Cells were transfected with control ( Ctrl ) , p97 or YOD1 siRNA oligos , treated with LLOMe , fixed after 12 h chase and stained for endogenous LC3 and K48 chains . Quantification of the number of K48 - positive vesicles per cell and number of LC3 - positive K48 vesicles per cell was determined . Data represent mean ± SD of three independent experiments . Scale bars , 10 µm .

O O O O O O B-MEASURED_VAR 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 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 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

E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation. Cells were transfected with control (Ctrl), p97 or YOD1 siRNA oligos, treated with LLOMe, fixed after 12 h chase and stained for endogenous LC3 and K48 chains. Quantification of the number of K48-positive vesicles per cell and number of LC3-positive K48 vesicles per cell was determined. Data represent mean ± SD of three independent experiments. Scale bars, 10 µm.


Immunoblot analysis of protein lysates from ARPE - 19 cells expressing TFEB - WT - FLAG or TFEB - S401A - FLAG treated with 200 μM NaAsO2 or 250 nM Torin - 1 for 1 h . Two different rabbit polyclonal antibodies raised against a TFEB - S401 phospho - specific peptide were tested .

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

Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-WT-FLAG or TFEB-S401A-FLAG treated with 200 μM NaAsO2 or 250 nM Torin-1 for 1 h. Two different rabbit polyclonal antibodies raised against a TFEB-S401 phospho-specific peptide were tested.


Immunoblot analysis of protein lysates from ARPE - 19 cells expressing TFEB - WT - FLAG , TFEB - S3A / R4A - FLAG or TFEB - R245 - 247A - FLAG treated with 200 μM NaAsO2 for 1 h . Immunoblots are representative of at least three independent experiments .

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

Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-WT-FLAG, TFEB-S3A/R4A-FLAG or TFEB-R245-247A-FLAG treated with 200 μM NaAsO2 for 1 h. Immunoblots are representative of at least three independent experiments.


Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2 for 1 h . Quantification of immunoblot data shown in ( A ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , and ( * * * * ) p < 0 . 0001 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

O O O O O O O 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

Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2 for 1 h. Quantification of immunoblot data shown in (A). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, and (****)p<0.0001 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG incubated with either 200 μM NaAsO2 for 1 h , EBSS for 4 h , 100 ng / ml EGF for the indicated times or 37 μM Anisomycin for 1 h . Before the addition of EGF or Anisomycin , cells were serum starved for 8 h .

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

Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG incubated with either 200 μM NaAsO2 for 1 h, EBSS for 4 h, 100 ng/ml EGF for the indicated times or 37 μM Anisomycin for 1 h. Before the addition of EGF or Anisomycin, cells were serum starved for 8 h.


Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG exposed to 30J / m2 of UV - C and allowed to recover in complete medium for the indicated times . Cells were incubated with p38 MAPK inhibitor ( 20 μM , SB203580 ) for 1 h before UV - C irradiation and allowed to recover for 30 min in the presence of the inhibitor .

O O O O O O O O O O O O O O O O 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 I-CONTROLLED_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

Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG exposed to 30J/m2 of UV-C and allowed to recover in complete medium for the indicated times. Cells were incubated with p38 MAPK inhibitor (20 μM, SB203580) for 1 h before UV-C irradiation and allowed to recover for 30 min in the presence of the inhibitor.


Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG depleted of either p38 MAPK ( α + β ) or JNK1 or JNK2 or JNK ( 1 + 2 ) and incubated with 200 μM NaAsO2 for 1 h . Immunoblots are representative of at least 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG depleted of either p38 MAPK (α+β) or JNK1 or JNK2 or JNK(1+2) and incubated with 200 μM NaAsO2 for 1 h. Immunoblots are representative of at least three independent experiments.


Immunoblot analysis of in vitro p38 MAPK kinase assay using GST - TFEB - PRD as substrate in the presence or absence of either recombinant human active p38α MAPK or ATP . Quantification of immunoblot data shown in ( F ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( * ) p < 0 . 05 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

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

Immunoblot analysis of in vitro p38 MAPK kinase assay using GST-TFEB-PRD as substrate in the presence or absence of either recombinant human active p38α MAPK or ATP. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Immunoblot analysis of protein lysates from Raw 264 . 7 cells stably expressing TFEB - WT - FLAG incubated with 1μg / ml LPS for the indicated times .

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

Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with 1μg/ml LPS for the indicated times.


Immunoblot analysis of protein lysates from Raw 264 . 7 cells stably expressing TFEB - WT - FLAG incubated with either 20 μM SB203580 or 250 nM Torin - 1 for 1 h prior to the addition of 1 μg / ml LPS for 30 min .

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

Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with either 20 μM SB203580 or 250 nM Torin-1 for 1 h prior to the addition of 1 μg/ml LPS for 30 min.


Quantification of immunoblot data shown in ( B ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , ( * ) p < 0 . 05 and ( * * ) p < 0 . 01 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

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

Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05 and (**)p<0.01 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Immunoblot analysis of protein lysates from THP1 cells incubated with 1 μg / ml LPS for the indicated times .

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

Immunoblot analysis of protein lysates from THP1 cells incubated with 1 μg/ml LPS for the indicated times.


Immunoblot analysis of protein lysates from THP1 cells incubated with either vehicle ( DMSO ) or 20 μM SB203580 for 1 h prior to the addition of 1μg / ml LPS for the indicated times .

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 B-CONTROLLED_VAR O O O O O

Immunoblot analysis of protein lysates from THP1 cells incubated with either vehicle (DMSO) or 20 μM SB203580 for 1 h prior to the addition of 1μg/ml LPS for the indicated times.


Immunoblot analysis of protein lysates from THP1 cells depleted of p38 MAPK ( α ) , p38 MAPK ( β ) or p38 MAPK ( α + β ) and incubated with 1 μg / ml LPS for 1 h . Immunoblots are representative of at least three independent experiments . Quantification of immunoblot data shown in ( F ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( * ) p < 0 . 05 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

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

Immunoblot analysis of protein lysates from THP1 cells depleted of p38 MAPK (α), p38 MAPK (β) or p38 MAPK (α+β) and incubated with 1 μg/ml LPS for 1 h. Immunoblots are representative of at least three independent experiments. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Immunoblot analysis of protein lysates from naïve THP1 - WT or TFEB - S401A knock - in ( clone M17 ) cells treated with 50 ng / ml PMA for the indicated times , PMA - differentiated THP1 ( Rested ) cells treated without or with 250 nM Torin - 1 for 1 h .

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

Immunoblot analysis of protein lysates from naïve THP1-WT or TFEB-S401A knock-in (clone M17) cells treated with 50 ng/ml PMA for the indicated times, PMA-differentiated THP1 (Rested) cells treated without or with 250 nM Torin-1 for 1 h.


Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells treated with 50 ng / ml PMA for the indicated times and PMA - differentiated THP1 ( Rested ) cells .

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

Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells treated with 50 ng/ml PMA for the indicated times and PMA-differentiated THP1 (Rested) cells.


Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA for the indicated times .

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 B-CONTROLLED_VAR O O O O O

Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for the indicated times.


Immunoblot analysis of proteins from nuclear fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA or 250 nM Torin - 1 for 1 h . Immunoblots are representative of at least three independent experiments .

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

Immunoblot analysis of proteins from nuclear fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA or 250 nM Torin-1 for 1 h. Immunoblots are representative of at least three independent experiments.


Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA for 6 h .

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 B-CONTROLLED_VAR O O O O

Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for 6 h.


Immunoblot analysis of protein lysates and cell culture medium from PMA - differentiated ( Rested ) THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells incubated with 0 . 1 μg / ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min . C - F . Quantification of immunoblot data shown in ( B ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 and ( * * * * ) p < 0 . 0001 from three independent experiments . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) .

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

Immunoblot analysis of protein lysates and cell culture medium from PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 0.1 μg/ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min. C-F. Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05, (**)p<0.01, (***)p< 0.001 and (****)p< 0.0001 from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).


Fluorescence confocal microscopy of PMA - differentiated ( Rested ) THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells incubated with 1 μg / ml LPS for 6 h prior to the detection of dead cells with LIVE / DEAD fixable blue dead cell stain . Scale bars , 10 μm . Quantification of the percentage of dead cells shown in ( G ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant and ( * * * * ) p < 0 . 0001 as compared to their corresponding untreated ( Control ) cells , with > 200 cells counted per trial from three independent experiments . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) .

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

Fluorescence confocal microscopy of PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 1 μg/ml LPS for 6 h prior to the detection of dead cells with LIVE/DEAD fixable blue dead cell stain. Scale bars, 10 μm. Quantification of the percentage of dead cells shown in (G). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant and (****)p< 0.0001 as compared to their corresponding untreated (Control) cells, with >200 cells counted per trial from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).


A . Blood - stage growth rate of concavin ( - ) parasites in comparison to wild type . Data points represent parasites growing in individual mice , n indicates total number . P - value is calculated using the Mann Whitney test . Shown is the mean ± SEM .

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

A. Blood-stage growth rate of concavin(-) parasites in comparison to wild type. Data points represent parasites growing in individual mice, n indicates total number. P-value is calculated using the Mann Whitney test. Shown is the mean ± SEM.


B . Gametocyte to ookinete conversion of 3 biological replicates . P - value calculated using the Mann Whitney test . Shown is the mean ± SEM . C . Average speed of moving ookinetes . Data points represent individual ookinetes , n indicates total number of cells from 3 biological replicates . P - values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison . D . Ookinete images of wild type and concavin ( - ) parasites revealing similar cell shapes . 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 O O O O 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

B. Gametocyte to ookinete conversion of 3 biological replicates. P-value calculated using the Mann Whitney test. Shown is the mean ± SEM. C. Average speed of moving ookinetes. Data points represent individual ookinetes, n indicates total number of cells from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison. D. Ookinete images of wild type and concavin(-) parasites revealing similar cell shapes. Scale bar 5 µm.


E . Oocyst development of concavin ( - ) parasites compared to wild type . Data points represent individual midguts observed between d12 - 17 post infection from 3 independent cage feeds . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison .

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

E. Oocyst development of concavin(-) parasites compared to wild type. Data points represent individual midguts observed between d12-17 post infection from 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison.


F . Mosquito infections resulted in deformed sporozoites in the salivary gland . Shown are example images of different sporozoites arranged to illustrate their rounding up over time . G . Quantification of sporozoite rounding up over time in the midgut ( MG ) the haemolymph ( HL ) and salivary gland ( SG ) at the indicated days post infection from three biological replicates . Sporozoites were classified as either normal , deformed or round as illustrated in F .

F. Mosquito infections resulted in deformed sporozoites in the salivary gland. Shown are example images of different sporozoites arranged to illustrate their rounding up over time. G. Quantification of sporozoite rounding up over time in the midgut (MG) the haemolymph (HL) and salivary gland (SG) at the indicated days post infection from three biological replicates. Sporozoites were classified as either normal, deformed or round as illustrated in F.


H . Average speed of salivary gland sporozoites . Data points represent individual sporozoites , n indicates total number from 3 biological replicates . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison 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

H. Average speed of salivary gland sporozoites. Data points represent individual sporozoites, n indicates total number from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


I . Selected trajectories of manually tracked sporozoites .

I. Selected trajectories of manually tracked sporozoites.


A . Oocyst development of concavin ( - ) parasites complemented with either the P . berghei gene or the P . falciparum orthologue fused to GFP . Data points represent individual midguts observed between d12 - 17 post infection of 3 independent cage feeds . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test .

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

A. Oocyst development of concavin(-) parasites complemented with either the P. berghei gene or the P. falciparum orthologue fused to GFP. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


B . Average speed of salivary gland sporozoites . Data points represent individual sporozoites of 3 independent biological replicates . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison 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

B. Average speed of salivary gland sporozoites. Data points represent individual sporozoites of 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


C . Sum table of mosquito infections of wild type , concavin ( - ) and complemented lines expressing either P . berghei ( Pb ) or P . falciparum ( Pf ) concavin - GFP . Numbers determined on d17 post mosquito infection . Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections . Note the difference of normally shaped and total sporozoites for concavin ( - ) parasites . Images show infection of salivary glands . Scale bar : 100

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

C. Sum table of mosquito infections of wild type, concavin(-) and complemented lines expressing either P. berghei (Pb) or P. falciparum (Pf) concavin-GFP. Numbers determined on d17 post mosquito infection. Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections. Note the difference of normally shaped and total sporozoites for concavin(-) parasites. Images show infection of salivary glands. Scale bar: 100 µm


D . Pb concavin - GFP and Pf concavin - GFP localization in blood and mosquito stages . Nuclei ( blue ) stained with Hoechst . Scale bar : 5 µm .

O O O B-MEASURED_VAR 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

D. Pb concavin-GFP and Pf concavin-GFP localization in blood and mosquito stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.


E . Localization of P . berghei concavin - GFP in liver stages . Nuclei ( blue ) stained with Hoechst . Scale bar : 5 µm .

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

E. Localization of P. berghei concavin-GFP in liver stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.


F . Immunofluorescence images of permeabilized and unpermeabilized concavin - GFP expressing salivary gland sporozoites stained with an anti - GFP antibody . Note that a GFP signal could only be detected after permeabilization , excluding concavin localization on the parasite surface as illustrated in the model . Scale bar : 5 µm .

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

F. Immunofluorescence images of permeabilized and unpermeabilized concavin-GFP expressing salivary gland sporozoites stained with an anti-GFP antibody. Note that a GFP signal could only be detected after permeabilization, excluding concavin localization on the parasite surface as illustrated in the model. Scale bar: 5 µm.


A , B . Time series of concavin - GFP ( A ) PhiL1 - GFP ( B ) sporozoites before and after bleaching and quantification of the fluorescence signal over time of 3 technical replicates . * indicates time of bleaching . Shown is the mean ± SD .

O O 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 O 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. Time series of concavin-GFP (A) PhiL1-GFP (B) sporozoites before and after bleaching and quantification of the fluorescence signal over time of 3 technical replicates. * indicates time of bleaching. Shown is the mean ± SD.


C . Super resolution ( STED ) imaging of PhiL1 - GFP and CSP as well as concavin - GFP and CSP . Cells were stained with an anti - GFP antibody in combination with Atto - 594 ( green ) in addition to an anti - CSP staining in combination with Atto - 647 . Images were deconvolved using the Richardson - Lucy algorithm . The distance between the 2 signal peaks was measured using the plot profile of the respective channels in Fiji . Data points represent distance in individual sporozoites at the center of the cell from 2 independent biological replicates . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test . Scale bar : 1 µm .

O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O 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. Super resolution (STED) imaging of PhiL1-GFP and CSP as well as concavin-GFP and CSP. Cells were stained with an anti-GFP antibody in combination with Atto-594 (green) in addition to an anti-CSP staining in combination with Atto-647. Images were deconvolved using the Richardson-Lucy algorithm. The distance between the 2 signal peaks was measured using the plot profile of the respective channels in Fiji. Data points represent distance in individual sporozoites at the center of the cell from 2 independent biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test. Scale bar: 1 µm.


D . Localization of PhiL1 - GFP ( green ) in concavin ( - ) parasites . Nuclei ( blue ) stained with Hoechst . Scale bar 5 µm . E . Localization of SiR - Tubulin ( red ) in concavin ( - ) parasites . Nuclei ( blue ) stained with Hoechst . Scale bar 5 µm . F . Localization of CSP ( red ) in concavin ( - ) parasites . Nuclei ( blue ) stained with Hoechst . Scale bar 5 µm . Data information : Asterisks in D - F indicate apical part of the sporozoite .

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 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 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 O O O O

D. Localization of PhiL1-GFP (green) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. E. Localization of SiR-Tubulin (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. F. Localization of CSP (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. Data information: Asterisks in D-F indicate apical part of the sporozoite.


B . Expression and localization of concavinC7A - GFP in blood stage parasites . Scale bar 5 µm .

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

B. Expression and localization of concavinC7A-GFP in blood stage parasites. Scale bar 5 µm.


C . Oocysts number in infected mosquitoes . Data points represent individual midguts observed between d12 - 17 post infection of 3 independent cage feeds . Shown is the mean ± SEM . P - value calculated using the 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

C. Oocysts number in infected mosquitoes. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-value calculated using the Mann Whitney test.


D . Quantification of concavinC7A - GFP cell shapes at the indicated days from midgut ( MG ) or salivary gland ( SG ) derived sporozoites . Numbers above bars indicate investigated sporozoites .

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

D. Quantification of concavinC7A-GFP cell shapes at the indicated days from midgut (MG) or salivary gland (SG) derived sporozoites. Numbers above bars indicate investigated sporozoites.


E . Average speed of salivary gland sporozoites . Data points represent individual sporozoites from 3 independent biological replicates . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test .

E. Average speed of salivary gland sporozoites. Data points represent individual sporozoites from 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


F . Localization of concavinC7A - GFP in normal and deformed sporozoites ( arrowheads point to deformations ) . Scale bar : 5 µm .

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

F. Localization of concavinC7A-GFP in normal and deformed sporozoites (arrowheads point to deformations). Scale bar: 5 µm.


A . Growth curve of blood stage parasites in C57BL / 6 mice infected by the bite of 10 mosquitoes ( left ) or 10 . 000 sporozoites intra venously ( right ) at two different times post mosquito infection as indicated . Shown is the mean ± SEM . Mice infected with Concavin ( - ) parasites by bite represent 2 independent biological replicates with 3 mice each . Wild type bite back and Concavin ( - ) intra venous injections represent one biological replicate using 3 or 4 mice 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 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

A. Growth curve of blood stage parasites in C57BL/6 mice infected by the bite of 10 mosquitoes (left) or 10.000 sporozoites intra venously (right) at two different times post mosquito infection as indicated. Shown is the mean ± SEM. Mice infected with Concavin(-) parasites by bite represent 2 independent biological replicates with 3 mice each. Wild type bite back and Concavin(-) intra venous injections represent one biological replicate using 3 or 4 mice respectively.


C . Liver cell invasion assay of concavin - gfp and concavin ( - ) parasites . Parasites are positively stained for CSP in case they remain extracellular . Both , normal and deformed parasites were detected intracellularly . Graph shows quantification of CSP positive ( red , hence extracellular ) and negative ( grey , hence intracellular ) sporozoites . Small graph shows the percentage of deformed and normally shaped intracellular concavin ( - ) sporozoites . Data points represent the 4 individual biological replicates with n indicating the numbers of sporozoites observed . Shown is the mean ± SEM . P - values are calculated using the Mann Whitney test . Scale bar : 5 µm .

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

C. Liver cell invasion assay of concavin-gfp and concavin(-) parasites. Parasites are positively stained for CSP in case they remain extracellular. Both, normal and deformed parasites were detected intracellularly. Graph shows quantification of CSP positive (red, hence extracellular) and negative (grey, hence intracellular) sporozoites. Small graph shows the percentage of deformed and normally shaped intracellular concavin(-) sporozoites. Data points represent the 4 individual biological replicates with n indicating the numbers of sporozoites observed. Shown is the mean ± SEM. P-values are calculated using the Mann Whitney test. Scale bar: 5 µm.


D . Liver - stage development of concavin ( - ) parasites compared to wildtype , both expressing cytoplasmic GFP ( white ) . Parasite size was measured 24h and 48h post infection . Data points represent individual parasites from 4 independent biological replicates . Shown is the mean ± SEM . P - values calculated using the Mann Whitney test . Scale bar : 5 µm .

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

D. Liver-stage development of concavin(-) parasites compared to wildtype, both expressing cytoplasmic GFP (white). Parasite size was measured 24h and 48h post infection. Data points represent individual parasites from 4 independent biological replicates. Shown is the mean ± SEM. P-values calculated using the Mann Whitney test. Scale bar: 5 µm.


E . Sporozoite ejection of immobilized concavin ( - ) infected mosquitoes on glass slides and quantification of ejected sporozoites from 20 mosquitoes . * indicates individual sporozoites in the ejected saliva . 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

E. Sporozoite ejection of immobilized concavin(-) infected mosquitoes on glass slides and quantification of ejected sporozoites from 20 mosquitoes. * indicates individual sporozoites in the ejected saliva. Scale bar: 10 µm.


F , G . Only normally shaped concavin ( - ) sporozoites released by salivary glands move on helical paths ( arrows ) through polyacrylamide gels that mimic the skin ( F ) . Quantification of two individual experiments Scale bar 10 µm . ( G ) : only normal shaped sporozoites were able to migrate through the gel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G. Only normally shaped concavin(-) sporozoites released by salivary glands move on helical paths (arrows) through polyacrylamide gels that mimic the skin (F). Quantification of two individual experiments Scale bar 10 µm. (G): only normal shaped sporozoites were able to migrate through the gel.


A . Percentage of mosquitoes depositing WT or concavin ( - ) sporozoites in the skin of a mouse during a bite ( left ) and sporozoites deposited during a mosquito bite ( right ) . P - values are calculated using the Fishers exact test ( left ) and the Mann Whitney test ( right ) . Bite transmission experiments were repeated over 3 independent mosquito infections for the KO and 2 independent mosquito infections for the WT . For each mosquito infection 2 - 3 mice were imaged , for a total of 3 - 5 ears .

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

A. Percentage of mosquitoes depositing WT or concavin(-) sporozoites in the skin of a mouse during a bite (left) and sporozoites deposited during a mosquito bite (right). P-values are calculated using the Fishers exact test (left) and the Mann Whitney test (right). Bite transmission experiments were repeated over 3 independent mosquito infections for the KO and 2 independent mosquito infections for the WT. For each mosquito infection 2-3 mice were imaged, for a total of 3-5 ears.


B . Maximum fluorescence intensity projections encoded by color for time from movies showing migrating sporozoites after mosquito - bite transmission . Graphs and camembert diagrams below show fraction of motile and immotile sporozoites after 10 , 20 and 30 minutes of recording , numbers analysed as well as sporozoite speed . Pooled data from 4 - 5 WT or 5 - 8 concavin ( - ) bite sites per time point . Scale bar : 50 µm . P - values are calculated using the 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 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

B. Maximum fluorescence intensity projections encoded by color for time from movies showing migrating sporozoites after mosquito-bite transmission. Graphs and camembert diagrams below show fraction of motile and immotile sporozoites after 10, 20 and 30 minutes of recording, numbers analysed as well as sporozoite speed. Pooled data from 4-5 WT or 5-8 concavin (-) bite sites per time point. Scale bar: 50 µm. P-values are calculated using the Mann Whitney test.


C . Motile WT or concavin ( - ) sporozoites entering blood vessels during the first hour following micro - injection . Number above bars shows numbers of sporozoites observed . Pooled data from four WT or five concavin ( - ) experiments . P - values are calculated using the Fishers exact test .

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

C. Motile WT or concavin(-) sporozoites entering blood vessels during the first hour following micro-injection. Number above bars shows numbers of sporozoites observed. Pooled data from four WT or five concavin(-) experiments. P-values are calculated using the Fishers exact test.


D , E ( D ) Deformation and ( E ) disintegration of concavin ( - ) sporozoites migrating in the skin . Individual images corresponding to the frames shown on the right are indicated in distinct colours in the maximum projection ( left ) of 13 - min movies . Arrowheads indicate constrictions of the parasites . Graphs below time - lapse show that deformation and disintegration are preceded by a decrease in speed . Color of dots correspond to the time - points displayed in the time - lapse images . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (D) Deformation and (E) disintegration of concavin(-) sporozoites migrating in the skin. Individual images corresponding to the frames shown on the right are indicated in distinct colours in the maximum projection (left) of 13-min movies. Arrowheads indicate constrictions of the parasites. Graphs below time-lapse show that deformation and disintegration are preceded by a decrease in speed. Color of dots correspond to the time-points displayed in the time-lapse images. Scale bars: 10 µm.


F . Percentage of deformation and disintegration events observed in 54 WT and 57 concavin ( - ) sporozoites that were tracked for at least 10 min . Pooled data from 5 WT or 10 concavin ( - ) bite sites .

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 B-CONTROLLED_VAR O O O O O O

F. Percentage of deformation and disintegration events observed in 54 WT and 57 concavin(-) sporozoites that were tracked for at least 10 min. Pooled data from 5 WT or 10 concavin(-) bite sites.


B , Same as in A , with the exception that cells expressing flag - praja2rm or praja2 deletion mutants ( praja21 - 530 , praja21 - 630 and praja2Δ530 - 630 ) were included in the analysis .

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

B, Same as in A, with the exception that cells expressing flag-praja2rm or praja2 deletion mutants (praja21-530, praja21-630 and praja2Δ530-630) were included in the analysis.


B . MST signal ( normalized fluorescence ) of P1 ( red curve ) , P2 ( green curve ) and P3 ( cyan curve ) plotted against TBC1D31 , at increasing concentrations of peptides . The threading modelled structure of the overlapping binding segment of praja2 ( praja2550 - 570 ) is 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

B. MST signal (normalized fluorescence) of P1 (red curve), P2 (green curve) and P3 (cyan curve) plotted against TBC1D31, at increasing concentrations of peptides. The threading modelled structure of the overlapping binding segment of praja2 (praja2550-570) is shown.


F . MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides : wild - type ( red curve ) , R948A - R951A ( violet curve ) and R957 - R959D - H960A ( orange curve ) .

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

F. MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides: wild-type (red curve), R948A-R951A (violet curve) and R957-R959D-H960A (orange curve).


D . Total lysates from mouse brain were immunoprecipitated with anti - TBC1D31 antibodies or with control IgG . The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies .

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

D. Total lysates from mouse brain were immunoprecipitated with anti-TBC1D31 antibodies or with control IgG. The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies.


E . Same as in C , with the exception that cells were co - transfected with flag - OFD1 vector and with control siRNA ( siCNT ) or siRNA targeting praja2 . F . Quantitative analysis of the experiments shown in E . A mean value ± SD of three independent experiments is shown . Student ' s t test , * p < 0 , 05 . G

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 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

E. Same as in C, with the exception that cells were co-transfected with flag-OFD1 vector and with control siRNA (siCNT) or siRNA targeting praja2. F. Quantitative analysis of the experiments shown in E. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05. G


C . HEK293 cells transfected with siRNAs were serum deprived , pre - treated with cycloheximide and stimulated with FSK at different time point . Lysates were immunoblotted for endogenous praja2 and OFD1 . Hsp90 was used as loading control . D . Quantitative analysis of the experiments shown in C . A mean value ± SD of three independent experiments is shown . Student ' s t test * p < 0 , 05 ; * * < 0 , 01 versus sipraja2 ( + FSK ) and basal values ( 0 ) . E

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 B-MEASURED_VAR 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 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

C. HEK293 cells transfected with siRNAs were serum deprived, pre-treated with cycloheximide and stimulated with FSK at different time point. Lysates were immunoblotted for endogenous praja2 and OFD1. Hsp90 was used as loading control. D. Quantitative analysis of the experiments shown in C. A mean value ± SD of three independent experiments is shown. Student's t test * p<0,05; **<0,01 versus sipraja2 (+FSK) and basal values (0). E


H . Immunoblot analysis of flag - OFD1 or flag - E97G mutant in HEK293 cells stimulated with FSK as in C . I . Quantitative analysis of the experiments shown in H . A mean value ± SD of three independent experiments is shown . Student ' s t test , * p < 0 , 05 versus E97G mutant ( + FSK ) and basal values ( 0 ) .

O O O O O O O B-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 O O O O O O 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

H. Immunoblot analysis of flag-OFD1 or flag-E97G mutant in HEK293 cells stimulated with FSK as in C. I. Quantitative analysis of the experiments shown in H. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05 versus E97G mutant (+FSK) and basal values (0).


A . siRNA transfected HEK293 cells were serum deprived and stained for acetylated - tubulin , TBC1D31 and DRAQ5 . B . Quantitative analysis of the experiments shown in A . A mean value ± SD of four independent experiments is shown . Student ' s t test * p < 0 , 05 . C

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

A. siRNA transfected HEK293 cells were serum deprived and stained for acetylated-tubulin, TBC1D31 and DRAQ5. B. Quantitative analysis of the experiments shown in A. A mean value ± SD of four independent experiments is shown. Student's t test *p< 0,05. C


C . HEK293 cells expressing flag - OFD1 or flag - S735A were serum deprived and subjected to staining analysis for acetylated α - tubulin , flag and DRAQ5 . Images were collected by super - resolution microscopy ( upper panels ) . Where indicated , flag - OFD1 or flag - S735A was co - transfected with cilia - APEX - GFP vector ( middle panels ) . NIH3T3 expressing flag - OFD1 or flag - S735A were stained with anti - ARL13B and anti - flag antibodies . D , E . Statistical analysis of the experiments shown in D . A mean value ± SD of three independent experiments is shown . For each experimental group , a minimum of 40 cilia were analyzed . Student ' s t test p * * * < 0 , 001 . F

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 O 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. HEK293 cells expressing flag-OFD1 or flag-S735A were serum deprived and subjected to staining analysis for acetylated α-tubulin, flag and DRAQ5. Images were collected by super-resolution microscopy (upper panels). Where indicated, flag-OFD1 or flag-S735A was co-transfected with cilia-APEX-GFP vector (middle panels). NIH3T3 expressing flag-OFD1 or flag-S735A were stained with anti-ARL13B and anti-flag antibodies. D, E. Statistical analysis of the experiments shown in D. A mean value ± SD of three independent experiments is shown. For each experimental group, a minimum of 40 cilia were analyzed. Student's t test p***<0,001. F


F . HEK293 cells transiently transfected with flag - OFD1 or flag - S735A were serum deprived , treated with FSK ( 6 hours ) and stained for flag , acetylated - tubulin and DRAQ5 . G . Statistical analysis of the experiments shown in F . A mean value ± SD of three independent experiments is shown . Cells analysed for each experiment : 300 for NT , 80 for flag - OFD1 and 80 for flag - S735A . Student ' s t test , p * < 0 , 05 , p * * < 0 , 01 . H

O O O O O O O O O B-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 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 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

F. HEK293 cells transiently transfected with flag-OFD1 or flag-S735A were serum deprived, treated with FSK (6 hours) and stained for flag, acetylated-tubulin and DRAQ5. G. Statistical analysis of the experiments shown in F. A mean value ± SD of three independent experiments is shown. Cells analysed for each experiment: 300 for NT, 80 for flag-OFD1 and 80 for flag-S735A. Student's t test, p*<0,05, p**<0,01. H


B . Confocal images of cilia of the neural tube cells in the wild - type , Ol - TBC1D31 KD , Ol - TBC1D31 KD + hTBC1D31 , wild - type hOFD1 , Ol - TBC1D31 KD + wild - type hOFD1 , hOFD1S735A , Ol - TBC1D31 KD + hOFD1S735A , Ol - TBC1D31 KD + hOFD1S735D and Ol - TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti - acetylated α - tubulin antibody ( green ) and DAPI ( blue ) .

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

B. Confocal images of cilia of the neural tube cells in the wild-type, Ol-TBC1D31 KD, Ol-TBC1D31 KD + hTBC1D31, wild-type hOFD1, Ol-TBC1D31 KD + wild-type hOFD1, hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735D and Ol-TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti-acetylated α-tubulin antibody (green) and DAPI (blue).


A - E Echocardiographic analysis of ( A ) posterior wall thickness , ( B ) septum thickness , ( C ) left ventricle end diastolic diameter , ( D ) fractional area change and ( E ) ejection fraction . n = 8 per genotype .

O O O O O O O O O O O O O O O O O O O O O O O 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-E Echocardiographic analysis of (A) posterior wall thickness, (B) septum thickness, (C) left ventricle end diastolic diameter, (D) fractional area change and (E) ejection fraction. n= 8 per genotype.


F Comparison of TAZ gene expression , normalized to S12 , in heart , liver , and kidney , analyzed in triplicates by quantitative PCR . n = 5 per genotype . G TAZ gene expression in percent . Wild - type is set to 100 % .

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

F Comparison of TAZ gene expression, normalized to S12, in heart, liver, and kidney, analyzed in triplicates by quantitative PCR. n= 5 per genotype.G TAZ gene expression in percent. Wild-type is set to 100%.


H , I Lipid species profiles of cardiolipin ( H ) and monolysocardiolipin ( I ) in isolated cardiac mitochondria analyzed by mass spectrometry . n = 4 per genotype .

O O O O O O O B-MEASURED_VAR 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

H, I Lipid species profiles of cardiolipin (H) and monolysocardiolipin (I) in isolated cardiac mitochondria analyzed by mass spectrometry. n= 4 per genotype.


J Comparison of ratios of total monolysocardiolipin and cardiolipin content in indicated tissues .

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

J Comparison of ratios of total monolysocardiolipin and cardiolipin content in indicated tissues.


A Periodic measurement of the oxygen consumption rate ( OCR ) of mitochondria , isolated from 5 mice per genotype , before and after the administration of the indicated compounds ( left ) . Quantification of OCR in independent experiments , n = 8 per genotype ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Periodic measurement of the oxygen consumption rate (OCR) of mitochondria, isolated from 5 mice per genotype, before and after the administration of the indicated compounds (left). Quantification of OCR in independent experiments, n= 8 per genotype (right).


B OCR rates of cardiac mitochondria after administration of pyruvate , malate and ADP , n = 7 per genotype .

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

B OCR rates of cardiac mitochondria after administration of pyruvate, malate and ADP, n= 7 per genotype.


A Mitochondrial membranes , isolated from cardiac tissue , were solubilized in 1 % digitonin and analyzed by BN - PAGE and Western - blotting with the indicated 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

A Mitochondrial membranes, isolated from cardiac tissue, were solubilized in 1% digitonin and analyzed by BN-PAGE and Western- blotting with the indicated antibodies.


B Cardiac mitochondria were prepared as in ( A ) and analyzed on a large pore BN - gel for better resolution of supercomplexes .

O 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 Cardiac mitochondria were prepared as in (A) and analyzed on a large pore BN-gel for better resolution of supercomplexes.


C Cardiac mitochondria were solubilized in 0 . 4 % DDM and complexes were separated by BN - PAGE and analyzed by Western - blotting with the indicated antibodies .

C Cardiac mitochondria were solubilized in 0.4% DDM and complexes were separated by BN-PAGE and analyzed by Western- blotting with the indicated antibodies.


D Mitochondria were isolated from kidney and liver and analyzed as in ( A ) .

D Mitochondria were isolated from kidney and liver and analyzed as in (A).


E Quantification of ROS production over time in cardiac mitochondria using the ROS - specific fluorescence - based sensor H2DCFDA . One representative measurement is shown . Analyses were performed in triplicates and confirmed for three animals per genotype . Significance was calculated using Student ' s T - TEST . sh , shTAZ .

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

E Quantification of ROS production over time in cardiac mitochondria using the ROS-specific fluorescence-based sensor H2DCFDA. One representative measurement is shown. Analyses were performed in triplicates and confirmed for three animals per genotype. Significance was calculated using Student's T-TEST. sh, shTAZ.


A Enzymatic activity of ubiquinone - cytochrome c oxidase in mitochondria isolated from cardiac tissue . The ubiquinone analogue decylubiquinone was used in this assay . Data were standardized to malate - dehydrogenase activity and presented as percent of wild - type , n = 4 per genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Enzymatic activity of ubiquinone-cytochrome c oxidase in mitochondria isolated from cardiac tissue. The ubiquinone analogue decylubiquinone was used in this assay. Data were standardized to malate-dehydrogenase activity and presented as percent of wild-type, n= 4 per genotype.


B Cytochrome c reductase activity , standardized to malate - dehydrogenase activity in cardiac mitochondria , were plotted as % of wild - type , n = 3 per genotype .

O O O O O O O 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 Cytochrome c reductase activity, standardized to malate-dehydrogenase activity in cardiac mitochondria, were plotted as % of wild-type, n= 3 per genotype.


C Protein levels of Cox1 and Rieske compared to VDAC3 in three different dilutions of isolated mitochondria , separated on SDS gel and visualized by Western - blotting .

O O O O B-MEASURED_VAR 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

C Protein levels of Cox1 and Rieske compared to VDAC3 in three different dilutions of isolated mitochondria, separated on SDS gel and visualized by Western-blotting.


D Respiratory chain complexes in cardiac mitochondria were separated on BN - PAGE and stained for activity of complex I , II , IV and F1FO - ATPase . sh , shTAZ ; Q , ubiquinone .

O 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 I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

D Respiratory chain complexes in cardiac mitochondria were separated on BN-PAGE and stained for activity of complex I, II, IV and F1FO- ATPase. sh, shTAZ; Q, ubiquinone.


A Enzymatic activity of the succinate dehydrogenase in isolated cardiac mitochondria from 3 animals per genotype .

A Enzymatic activity of the succinate dehydrogenase in isolated cardiac mitochondria from 3 animals per genotype.


B Mitochondrial membranes were solubilized in 1 % digitonin and analyzed by BN - PAGE and Western - blotting using antibodies against SDH components SDHA and SDHB ( upper panel ) . For a loading control , samples were separated on SDS gel and analyzed by Western - blotting ( lower panel ) .

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 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

B Mitochondrial membranes were solubilized in 1% digitonin and analyzed by BN-PAGE and Western-blotting using antibodies against SDH components SDHA and SDHB (upper panel). For a loading control, samples were separated on SDS gel and analyzed by Western-blotting (lower panel).


C Steady state protein levels analyzed by SDS gel separation of indicated amounts of mitochondrial protein and subsequent Western - blotting with antibodies against SDHA and VDAC3 or visualizing of Flavin fluorescence after excitation with 488 nm . Lower panel : quantification of signal ratio SDHA / VDAC3 in shTAZ and control ( set to 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 B-MEASURED_VAR 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

C Steady state protein levels analyzed by SDS gel separation of indicated amounts of mitochondrial protein and subsequent Western- blotting with antibodies against SDHA and VDAC3 or visualizing of Flavin fluorescence after excitation with 488 nm. Lower panel: quantification of signal ratio SDHA/VDAC3 in shTAZ and control (set to 100%).


D Analysis of gene expression in cardiac tissue analyzed in ( C ) by qPCR using primers against indicated mRNA . Data of one representative measurement , normalized to b - actin with WT set to 100 % are shown . Identical results were obtained in further experiments with two different pairs of animals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Analysis of gene expression in cardiac tissue analyzed in (C) by qPCR using primers against indicated mRNA. Data of one representative measurement, normalized to b-actin with WT set to 100% are shown. Identical results were obtained in further experiments with two different pairs of animals.


E Upper panel : SDH complex of cardiac , liver and kidney were analyzed by BN - PAGE as in ( B ) ; lower panel : the same samples were analyzed by SDS gel and Western - blotting .

O O O O O O O O O O O O O O O O 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 Upper panel: SDH complex of cardiac, liver and kidney were analyzed by BN-PAGE as in (B); lower panel: the same samples were analyzed by SDS gel and Western-blotting.