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

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

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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 . co - expressing LAMP1 - RFP GFP - p97 were treated as in ( A ) stained with pan - ubiquitin antibody FK2 ( Ub ) . of showing recruitment of p97 in three independent experiments ( mean ± SD ) .

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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 ) . of showing recruitment of p97 in three independent experiments ( mean ± SD ) .

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

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

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

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

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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 . transfected with control ( Ctrl ) or p97 siRNA oligos were treated as in ( G ) . of 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 .

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

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

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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 lines described in ( B ) were treated with LLOMe for 1 h chased for indicated time . LC3 GAPDH were analyzed by immunoblotting . is shown in Figure EV2B .

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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 skeletal muscle . biopsies immunostained for Gal3 caveolin - 3 ( Cav3 ) . from 4 random 10X fields for each biopsy means for controls , respectively . carry p97 mutations R155H ( # 1 - 3 ) or R93C for ( # 4 ) . # 2 # 3 are cousins . Scale bar , 100 µm .

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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 were transfected with indicated siRNA oligos LLOMe - treated for 1 h . were chased for 12 h , fixed stained for Gal3 . See Figure EV3 for depletion efficiency . B of of with more than three Gal3 vesicles per for each condition . This threshold was chosen so that less than 1 % of untreated were considered positive . Data represent mean ± of three independent experiments with ≥ 30 quantified per condition . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - sided Welch ' s t - ) .

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

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

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

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

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

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

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

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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 GFP - p97 - EQ , 2 h after LLOMe washout co - stained with indicated antibodies .

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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 - K48 - ubiquitination . Control - depleted were fixed at indicated time points after LLOMe washout stained with chain - specific antibodies LAMP1 . Automated 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 persistence of K48 chains on damaged lysosomes . Experiment as in ( A ) in treated with p97 or YOD1 siRNA oligos .

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

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

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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 indicated purified isolated . Co - isolated ( pulldown ) were detected in SDS - gels . Note binding of p97 YOD1 to Ub - GST only in combination .

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

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

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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 S2 site . LLOMe - treated expressing GFP - YOD1 - CS or double mutant GFP - YOD1 - CS - MutS2 were chased for 2 h stained for K48 chains LAMP1 . of GFP signal on K48 vesicles to GFP signal in whole was determined .

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

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

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

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

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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 . expressing GFP - YOD1 - CS were treated as in ( A ) stained for LAMP1 p62 or LC3 . of GFP - YOD1 vesicles positive for p62 or LC3 was determined ( mean ± SD ) .

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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 . were transfected with control ( Ctrl ) , p97 or YOD1 siRNA oligos , treated with LLOMe , fixed after 12 h chase stained for endogenous LC3 K48 chains . of number of K48 - positive vesicles per number of LC3 - positive K48 vesicles per was determined . Data represent mean ± SD of three independent experiments . Scale bars , 10 µm .

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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 of lysates from ARPE - 19 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 .

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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 of lysates from ARPE - 19 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 .

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

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

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

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

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

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

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

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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.


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

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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 of lysates from THP1 incubated with 1 μg / ml LPS for indicated times .

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


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

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

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

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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 of from fractions from naïve THP1 - WT or TFEB - S401A knock - ins ( clones I11 M17 ) treated with 50 ng / ml PMA for indicated times PMA - differentiated THP1 ( Rested ) .

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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 of from fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) treated with 50 ng / ml PMA for indicated times .

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

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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 of from fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) treated with 50 ng / ml PMA for 6 h .

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

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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).


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

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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 ( - ) in comparison to wild type . Data points represent growing in mice , n indicates total number . P - value is calculated using Mann Whitney . Shown is mean ± .

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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 Mann Whitney . Shown is mean ± . C . Average speed of moving ookinetes . Data points represent ookinetes , n indicates total number of from 3 biological replicates . P - values are calculated using Kruskal Wallis followed by Dunns multiple comparison . D . Ookinete of wild type concavin ( - ) revealing similar shapes . Scale bar 5 µm .

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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 ( - ) compared to wild type . Data points represent midguts observed between d12 - 17 post infection from 3 independent cage feeds . Shown is mean ± . P - values are calculated using Kruskal Wallis followed by Dunns multiple comparison .

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

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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 sporozoites . Data points represent sporozoites , n indicates total number from 3 biological replicates . P - values are calculated using Kruskal Wallis followed by Dunns multiple comparison .

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

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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 sporozoites . Data points represent sporozoites of 3 independent biological replicates . Shown is mean ± . P - values are calculated using Kruskal Wallis followed by Dunns multiple comparison .

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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 ( - ) 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 difference of normally shaped total sporozoites for concavin ( - ) . show infection of salivary . Scale bar : 100

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

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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 .

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


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

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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 after bleaching of signal over time of 3 technical replicates . * indicates time of bleaching . Shown is mean ± SD .

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

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

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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 localization of concavinC7A - GFP in blood stage . Scale bar 5 µm .

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


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

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

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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 sporozoites . Data points represent sporozoites from 3 independent biological replicates . Shown is mean ± . P - values are calculated using Kruskal Wallis followed by Dunns multiple comparison .

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 deformed sporozoites ( arrowheads point to deformations ) . Scale bar : 5 µm .

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


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

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

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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 ( - ) compared to wildtype , both expressing GFP ( ) . was measured 24h 48h post infection . Data points represent from 4 independent biological replicates . Shown is mean ± . P - values calculated using Mann Whitney . Scale bar : 5 µm .

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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 of ejected sporozoites from 20 mosquitoes . * indicates sporozoites in ejected saliva . Scale bar : 10 µm .

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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 move on helical paths ( arrows ) through polyacrylamide gels that mimic skin ( F ) . of two experiments Scale bar 10 µm . ( G ) : only normal shaped sporozoites were able to migrate through gel .

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

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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 projections encoded by color for time from movies showing migrating sporozoites after mosquito - bite transmission . Graphs camembert diagrams below show fraction of motile immotile sporozoites after 10 , 20 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 Mann Whitney .

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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 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 Fishers exact .

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

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

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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 exception that expressing flag - praja2rm or praja2 deletion mutants ( praja21 - 530 , praja21 - 630 praja2Δ530 - 630 ) were included in .

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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 ) of P1 ( red curve ) , P2 ( green curve ) P3 ( cyan curve ) plotted against TBC1D31 , at increasing concentrations of peptides . threading modelled structure of overlapping binding segment of praja2 ( praja2550 - 570 ) is shown .

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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 ) R957 - R959D - H960A ( orange curve ) .

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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 . precipitates an aliquot of lysates were immunoblotted with indicated antibodies .

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

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

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

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

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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 expressing flag - OFD1 or flag - S735A were serum deprived subjected to for acetylated α - tubulin , flag DRAQ5 . 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 anti - flag antibodies . D , E . Statistical of 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 p * * * < 0 , 001 . F

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

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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 of cilia of neural tube in 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 Ol - TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti - acetylated α - tubulin antibody ( green ) DAPI ( blue ) .

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

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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 , kidney , analyzed in triplicates by quantitative PCR . n = 5 per genotype . G TAZ gene expression in . Wild - type is set to 100 % .

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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 ) monolysocardiolipin ( I ) in isolated cardiac mitochondria analyzed by mass spectrometry . n = 4 per genotype .

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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 cardiolipin content in indicated tissues .

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


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

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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 ADP , n = 7 per genotype .

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 analyzed by BN - PAGE Western - blotting with indicated antibodies .

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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 ) analyzed on a large pore BN - gel for better resolution of supercomplexes .

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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 complexes were separated by BN - PAGE analyzed by Western - blotting with 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 liver analyzed as in ( A ) .

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


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

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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 . ubiquinone analogue decylubiquinone was used in this . Data were standardized to malate - dehydrogenase activity presented as of wild - type , n = 4 per genotype .

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 .

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


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

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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 stained for activity of I , II , IV F1FO - ATPase . sh , shTAZ ; Q , ubiquinone .

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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 succinate dehydrogenase in isolated cardiac mitochondria from 3 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 analyzed by BN - PAGE Western - blotting using antibodies against SDH components SDHA SDHB ( upper panel ) . For a loading control , samples were separated on SDS gel analyzed by Western - blotting ( lower panel ) .

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

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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 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 .

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

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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.


F , G of enzymatic activity of succinate dehydrogenase related to malate dehydrogenase activity in mitochondria isolated from kidney ( F ) liver ( G ) . Values of 3 per genotype are plotted as of wild - type . sh , shTAZ .

F, G Analysis of enzymatic activity of the succinate dehydrogenase related to the malate dehydrogenase activity in mitochondria isolated from kidney (F) and liver (G). Values of 3 animals per genotype are plotted as percent of wild-type. sh, shTAZ.


A Flow cytometry of cardiac troponinT ( cTNT ) expression was performed at day 60 post cardiac differentiation . 98 % of cTNT + cardiomyocytes ( CM ) were obtained after metabolic selection .

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A Flow cytometry analysis of cardiac troponinT (cTNT) expression was performed at day 60 post cardiac differentiation. 98% of cTNT+ cardiomyocytes (CM) were obtained after metabolic selection.


B RT - PCR showing expression of indicated genes in undifferentiated iPSCs differentiated iPSC - CMs of BTHS TAZ10 a healthy control .

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B RT-PCR showing the expression of indicated genes in undifferentiated iPSCs and differentiated iPSC-CMs of BTHS patient TAZ10 and a healthy control.


C Immunostaining of control ( left ) BTHS iPSC - CMs at day 60 post differentiation for cardiac troponin T ( cTNT ) Connexin 43 ( Cx43 ) . Scale bars : 50µm .

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C Immunostaining of control (left) and BTHS iPSC-CMs at day 60 post differentiation for cardiac troponin T (cTNT) and Connexin 43 (Cx43). Scale bars: 50µm.


D Quantitation of sarcomere organization . Left panel : Immunostaining of cTNT , MLC2a ( myosin chain ) a - actinin at day 60 post cardiac differentiation . Right panel : bar graphs at right panel represent sarcomere regularity from 3 independent experiments . Error bars represent standard deviation for comparison between control BTHS TAZ10 . Significance was analyzed using Student ' s T - * * P = 0 . 002 * * * P = 0 . 0006 . Scale bars : 50 µm .

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D Quantitation of sarcomere organization. Left panel: Immunostaining of cTNT, MLC2a (myosin light chain) and a-actinin at day 60 post cardiac differentiation. Right panel: The bar graphs at the right panel represent the sarcomere regularity from 3 independent experiments. Error bars represent standard deviation for the comparison between control and BTHS patient TAZ10. Significance was analyzed using Student's T-TEST **P = 0.002 and ***P = 0.0006. Scale bars: 50 µm.


E Measurement of surface areas . surface was measured in single cardiomyocytes ( n = at least 30 measurements each group from N = 3 independent experiments each group ) at day 60 post cardiac differentiation after immunostaining of sarcomeric a - actinin . Scale bars : 50 µm .

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E Measurement of cell surface areas. Cell surface area was measured in single cardiomyocytes (n = at least 30 measurements each group from N = 3 independent experiments each group) at day 60 post cardiac differentiation after immunostaining of sarcomeric a-actinin. Scale bars: 50 µm.


A Oxygen consumption rate ( OCR ) of cardiomyocytes from TAZ10 control at basal conditions after administration of indicated compounds .

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A Oxygen consumption rate (OCR) of cardiomyocytes from patient TAZ10 and control at basal conditions and after the administration of the indicated compounds.


B Mitochondrial membranes analyzed by BN - PAGE Western - blotting using indicated antibodies . For a loading control samples were separated on SDS gel analyzed by Western - blotting ( lower panel ) .

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B Mitochondrial membranes analyzed by BN-PAGE and Western-blotting using indicated antibodies. For a loading control samples were separated on SDS gel and analyzed by Western-blotting (lower panel).


C Western blot of SDHA compared to VDAC3 . of SDHA relative to VDAC3 in with control set to 100 % . Error bars indicate range of values of two independent experiments .

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C Western blot analysis of SDHA protein levels compared to VDAC3. Quantification of SDHA relative to VDAC3 in percent with control set to 100%. Error bars indicate range of values of two independent experiments.


D of SDHA gene expression in triplicates by qPCR in cardiomyocytes , analyzed in ( C ) .

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D Analysis of SDHA gene expression in triplicates by qPCR in cardiomyocytes, analyzed in (C).


D . Representative immunofluorescence of MEFs 48h after transgene induction stained for indicated reprogramming factors . Scale bar represents 50 μm ( n = 3 biological replicates ) .

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D. Representative immunofluorescence images of MEFs 48h after transgene induction stained for the indicated reprogramming factors. Scale bar represents 50 μm (n = 3 biological replicates).


E . Log fluorophore expression superimposed on Uniform Manifold Approximation Projection ( UMAP ) embedding of dataset . Shown are fluorophore expression ( gene exp . ) on a logarithmic ( ln ) scale . F . Log transgene expression superimposed on a UMAP embedding of dataset . Shown are transgene expression ( gene exp . ) on an a logarithmic ( ln ) scale .

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E. Log fluorophore expression superimposed on Uniform Manifold Approximation and Projection (UMAP) embedding of the dataset. Shown are fluorophore expression levels (gene exp.) on a logarithmic (ln) scale. F. Log transgene expression superimposed on a UMAP embedding of the dataset. Shown are transgene expression levels (gene exp.) on an a logarithmic (ln) scale.


G . UMAP embedding of scRNA - seq dataset colored by technical replicate . Orange : Replicate 1 , Blue : Replicate

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G. UMAP embedding of scRNA-seq dataset colored by technical replicate. Orange: Replicate 1, Blue: Replicate 2


H . cycle score superimposed on UMAP embedding of dataset . See unsupervised of single - - seq section in Methods for further details .

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H. Cell cycle score superimposed on UMAP embedding of the dataset. See unsupervised analysis of single-cell RNA-seq section in Methods for further details.


D . Log - normalized expression z - score of genes up - ( top panels ) downregulated ( bottom panels ) by Ascl1 ( left , blue violins ) MyoD1 ( right , yellow violins ) versus their scaled expression ( x - axis ) binned into 10 % intervals . For each box , centerline defines median , height of box is given by interquartile range ( IQR ) , whiskers are given by 1 . 5 * IQR outliers are given as points beyond minimum or maximum whisker .

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D. Log-normalized expression z-score of genes up- (top panels) and downregulated (bottom panels) by Ascl1 (left, blue violins) and MyoD1 (right, yellow violins) versus their scaled expression levels (x-axis) binned into 10% intervals. For each box, the centerline defines the median, the height of the box is given by the interquartile range (IQR), the whiskers are given by 1.5 * IQR and outliers are given as points beyond the minimum or maximum whisker.


E . Fate titration plot of Ascl1 MyoD1 double positive Shown are decision boundaries for indicated fates according to transcription factor . MyoD1 Ascl1 expression shown on x - y - axis are log - normalized expression values scaled into dynamic range of single - positive condition are colored according to their lineage endpoint as determined by CellRank .

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E. Fate titration plot of Ascl1 and MyoD1 double positive cells Shown are decision boundaries for indicated fates according to transcription factor levels. MyoD1 and Ascl1 expression shown on the x- and y-axis are log-normalized expression values scaled into the dynamic range of the single-positive condition Cells are colored according to their lineage endpoint as determined by CellRank.


F . Representative immunofluorescence of 3 days after transgene induction . were stained for reporter present on PiggyBac construct of reprogramming factor . Arrows indicate multinucleated . Scale bar represents 50 µm . mutAscl1 = mutant Ascl1 . G . of of with more than one nucleus among transfected within a given condition . Data points represent biological replicates ( n = 4 ) . For each box , centerline defines median , height of box is given by interquartile range ( IQR ) , whiskers are given by 1 . 5 * IQR outliers are given as points beyond minimum or maximum whisker . Pairwise comparisons performed with Mann - Whitney U , correction for multiple testing performed with Benjamini - Hochberg correction . * : p < 0 . 05 ( MyoD1 vs Ascl1 : p = 0 . 03 , MyoD1 vs mutAscl1 : p = 0 . 03 , MyoD1 vs Ascl1 & MyoD1 : p = 0 . 03 , MyoD1 vs mutAscl1 & MyoD1 : p = 0 . 03 , Ascl1 vs Ascl1 & MyoD1 : p = 0 . 03 , Ascl1 vs mutAscl1 & MyoD1 : p = 0 . 03 , mutAscl1 vs Ascl1 & MyoD1 : p = 0 . 03 , mutAscl1 vs mutAscl1 & MyoD1 : p = 0 .

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F. Representative immunofluorescence images of cells 3 days after transgene induction. Cells were stained for the fluorescent reporter present on the PiggyBac construct of the reprogramming factor. Arrows indicate multinucleated cells. Scale bar represents 50 µm. mutAscl1 = mutant Ascl1. G. Quantification of the percentage of cells with more than one nucleus among all transfected cells within a given condition. Data points represent biological replicates (n = 4). For each box, the centerline defines the median, the height of the box is given by the interquartile range (IQR), the whiskers are given by 1.5 * IQR and outliers are given as points beyond the minimum or maximum whisker. Pairwise comparisons performed with Mann-Whitney U test, correction for multiple testing performed with Benjamini-Hochberg correction. *: p < 0.05 (MyoD1 vs Ascl1: p = 0.03, MyoD1 vs mutAscl1: p = 0.03, MyoD1 vs Ascl1 & MyoD1: p = 0.03, MyoD1 vs mutAscl1 & MyoD1: p = 0.03, Ascl1 vs Ascl1 & MyoD1: p = 0.03, Ascl1 vs mutAscl1 & MyoD1: p = 0.03, mutAscl1 vs Ascl1 & MyoD1: p = 0.03, mutAscl1 vs mutAscl1 & MyoD1: p = 0.03


( A ) in vitro kinase assays were performed using purified recombinant kinases ( ULK1 ULK2 ) substrate ( ATG16L1 ) in presence of radiolabelled ATP . ULK ATG16L1 inputs were examined by western blot ( WB ) substrate phosphorylation was analyzed by autoradiography ( AR ) . Data information : Unless otherwise indicated experiments were performed three times .

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(A) in vitro kinase assays were performed using purified recombinant kinases (ULK1 and ULK2) and substrate (ATG16L1) in the presence of radiolabelled ATP. ULK and ATG16L1 inputs were examined by western blot (WB) and substrate phosphorylation was analyzed by autoradiography (AR). Data information: Unless otherwise indicated experiments were performed three times.


( B ) Full - or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase . ULK1 ATG16L1 inputs were examined by western blot target phosphorylation by autoradiography . Data information : Unless otherwise indicated experiments were performed three times .

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(B) Full-length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay. ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography. Data information: Unless otherwise indicated experiments were performed three times.


( D ) Full - or mutated HA - ATG16L1 was purified from mammalian subjected to an in vitro ULK1 kinase . Inputs were analysed by WB target phosphorylation by AR . Data information : Unless otherwise indicated experiments were performed three times .

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(D) Full-length or mutated HA-ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay. Inputs were analysed by WB and target phosphorylation by AR. Data information: Unless otherwise indicated experiments were performed three times.


( E ) HEK293A were transfected with wild - type or phospho - dead ATG16L1 in presence of wild - type or kinase - dead ULK1 . Phosphorylation of ATG16L1 ( S278 or S287 ) inputs were examined by WB . Data information : Unless otherwise indicated experiments were performed three times .

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(E) HEK293A cells were transfected with wild-type or phospho-dead ATG16L1 in the presence of wild-type or kinase-dead ULK1. Phosphorylation of ATG16L1 (S278 or S287) and inputs were examined by WB. Data information: Unless otherwise indicated experiments were performed three times.