A , B Immunoblot of indicated lines , starved for 6 hours ( HBSS ) as indicated , treated with MG132 or Baf A1 as indicated . Numbers below blots in A represent relative of bands in shown blots normalized against GAPDH loading control . In B , bars represent mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA significance displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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A, B Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with MG132 or Baf A1 as indicated. Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In B, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


C Immunoblot of HeLa transfected with indicated siRNAs .

C Immunoblot analysis of HeLa cells transfected with the indicated siRNAs.


D , E Immunoblot of HeLa transfected with indicated siRNAs treated with Baf A1 as indicated . In D , panels are from more than one western blot experiment but only a single GAPDH loading control is shown . In E , bars represent mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 ; ns is not significant .

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D, E Immunoblot analysis of HeLa cells transfected with the indicated siRNAs and treated with Baf A1 as indicated. In D, the panels are from more than one western blot experiment but only a single GAPDH loading control is shown. In E, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005; ns is not significant.


F HeLa KO CALCOCO1 were transiently co - transfected with EGFP - CALCOCO1 Myc - VAPA or Myc - VAPB , then immunostained with anti - Myc anti - LC3B antibodies . Arrows indicate dots of co - localization of three . Scale bars , 5 μm for large merged 1 μm for zoomed .

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F HeLa KO CALCOCO1 cells were transiently co-transfected with EGFP-CALCOCO1 and Myc-VAPA or Myc-VAPB, and then immunostained with anti-Myc and anti-LC3B antibodies. Arrows indicate dots of co-localization of all three proteins. Scale bars, 5 μm for large merged images and 1 μm for zoomed images.


A , B Immunoblot of HeLa WT HeLa CALCOCO1 KO . were starved for 6 hours ( HBSS ) as indicated treated with Baf A1 as indicated . Numbers below blots in A represent relative of bands in shown blots normalized against GAPDH loading control . In A , panels are collected from more than one western blot experiment but for clarity , only a single GAPDH loading control is shown . In B , bars represent mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison significance displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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A, B Immunoblot analysis of HeLa WT and HeLa CALCOCO1 KO cells. The cells were starved for 6 hours (HBSS) as indicated and treated with Baf A1 as indicated. Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In A, the panels are collected from more than one western blot experiment but for clarity, only a single GAPDH loading control is shown. In B, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


C , D Immunoblot of HeLa CALCOCO1 KO lines reconstituted with EGFP - CALCOCO1 . Expression of EGFP - CALCOCO1 was induced or not with tetracycline were treated with MG132 or Baf A1 as indicated . Numbers below blots in C represent relative of bands in shown blots normalized against actin loading control . In C , panels are collected from more than one western blot experiment but only a single actin loading control is shown . In D , bars represent mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed as in B significance displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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C, D Immunoblot analysis of HeLa CALCOCO1 KO cell lines reconstituted with EGFP-CALCOCO1. Expression of EGFP-CALCOCO1 was induced or not with tetracycline and the cells were treated with MG132 or Baf A1 as indicated. Numbers below the blots in C represent relative intensity of the bands in the shown blots normalized against actin loading control. In C, the panels are collected from more than one western blot experiment but only a single actin loading control is shown. In D, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed as in B and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


E HeLa CALCOCO1 KO lines reconstituted with EGFP - CALCOCO1 were treated with tetracycline or not to induce expression of EGFP - CALCOCO1 . Abundance of ER was quantified from widefield of endogenous RTN3 ( see Materials methods ) . Data are presented as mean ± sd of three independent experiments . Statistical comparison was analyzed as in B significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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E HeLa CALCOCO1 KO cell lines reconstituted with EGFP-CALCOCO1 were treated with tetracycline or not to induce expression of EGFP-CALCOCO1. Abundance of the ER was quantified from widefield fluorescence images of endogenous RTN3 staining (see Materials and methods). Data are presented as mean ± sd of three independent experiments. Statistical comparison was analyzed as in B and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


F , G Immunoblot of analyzed in C . bars in G represent mean ± sd of band intensities relative to loading control from three independent experiments quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison . Significance is displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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F, G Immunoblot analysis of cells analyzed in C. The bars in G represent the mean±sd of band intensities relative to the loading control from three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


H , I Immunoblot of HeLa CALCOCO1 KO stably expressing EGFP - CALCOCO1 in fed or starved conditions transfected with indicated VAPA / B siRNAs .

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H, I Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 in fed or starved conditions and transfected with the indicated VAPA/B siRNAs.


A Immunoblot of HeLa CALCOCO1 KO stably expressing EGFP - CALCOCO1 in fed or starved conditions treated as indicated with Baf A1 or PI3KC3 inhibitor SAR405 .

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A Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 in fed or starved conditions and treated as indicated with Baf A1 or PI3KC3 inhibitor SAR405.


B Immunoblot of HeLa CALCOCO1 KO stably expressing EGFP - CALCOCO1 mLIR + ∆ 623 - 691 for 24 hours or not , then treated as indicated .

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B Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 mLIR+∆623-691 for 24 hours or not, and then treated as indicated.


C , Representative confocal of HeLa CALCOCO1 KO transiently co - transfected with mCherry - CALCOCO1 , Myc - VAPA EGFP - LAMP1 , then treated as indicated before immunostaining with anti - Myc anti - . Arrows indicate co - localization . Scale bars in C , 5 μm for large merged 1 μm for zoomed .

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C, Representative confocal images of HeLa CALCOCO1 KO cells transiently co-transfected with mCherry-CALCOCO1, Myc-VAPA and EGFP-LAMP1, and then treated as indicated before immunostaining with anti-Myc anti-body. Arrows indicate co-localization. Scale bars in C, 5 μm for large merged images and 1 μm for zoomed images.


D , data are presented as mean ± sd . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison significance displayed as * * * p ˂ 0 . 001 ; ns is not significant , n = 3 .

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D, data are presented as mean ± sd. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001; ns is not significant, n=3.


A Representative electron of HeLa treated or not with 250 µM LLOMe for 5 min or 10 min before chemical fixation . were incubated for 4 h following washing steps overnight incubation with 15 nm gold nanoparticles conjugated to bovine serum albumin to visualize lysosomes ( black spots inside lysosomes are internalized gold nanoparticles ) before treatment with LLOMe processing of samples for electron microscopy . Arrows indicate areas with increased lysosome - ER contacts . B of electron showing an increase in contact sites between ER lysosomes following 250 µM LLOMe treatment for 5 or 10 minutes . Error bars denote + / - SD from n = 3 independent experiments , > 48 lysosomes were quantified per experiment for each condition .

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A Representative electron micrographs of HeLa cells treated or not with 250 µM LLOMe for 5 min or 10 min before chemical fixation. Cells were incubated for 4 h following washing steps and overnight incubation with 15 nm gold nanoparticles conjugated to bovine serum albumin to visualize lysosomes (black spots inside lysosomes are internalized gold nanoparticles) before treatment with LLOMe and processing of samples for electron microscopy. Arrows indicate areas with increased lysosome-ER contacts. B Quantification of electron micrographs showing an increase in membrane contact sites between ER and lysosomes following 250 µM LLOMe treatment for 5 or 10 minutes. Error bars denote +/- SD from n=3 independent experiments, >48 lysosomes were quantified per experiment for each condition.


C Representative electron of VAP double knockout HeLa that were either left untreated or incubated with 250 µM LLOMe for 5 min or 10 min before chemical fixation . were incubated with 15 nm gold nanoparticles conjugated to bovine serum albumin for 4 h to visualize lysosomes . After 4 h , gold nanoparticles were washed off were incubated overnight before treatment with LLOMe processing samples for electron microscopy ( same as in A ) . of electron showing no increase in contact sites between ER lysosomes following 250 µM LLOMe treatment for 5 or 10 minutes in VAP double knockout . Error bars denote + / - SD from n = 3 independent experiments , > 40 lysosomes were quantified per experiment for each condition .

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C Representative electron micrographs of VAP double knockout HeLa cells that were either left untreated or incubated with 250 µM LLOMe for 5 min or 10 min before chemical fixation. Cells were incubated with 15 nm gold nanoparticles conjugated to bovine serum albumin for 4 h to visualize lysosomes. After 4 h, gold nanoparticles were washed off and cells were incubated overnight before treatment with LLOMe and processing samples for electron microscopy (same as in A). Quantification of electron micrographs showing no increase in membrane contact sites between ER and lysosomes following 250 µM LLOMe treatment for 5 or 10 minutes in VAP double knockout cells. Error bars denote +/- SD from n=3 independent experiments, >40 lysosomes were quantified per experiment for each condition.


D Representative movie stills of a live - imaging experiment illustrating recruitment of transiently expressed eGFP - ORP1L to lysosomes following 250 µM LLOMe treatment . were pre - treated for 30 min with 75 nM Lysotracker Deep Red to monitor lysosome damage ( judged by decreased number of Lysotracker spots after LLOMe treatment ) . graph represents of eGFP - ORP1L foci per after LLOMe treatment . Error bars denote + / - from n = 3 independent live - imaging experiments , > 25 were analyzed per experiment .

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D Representative movie stills of a live-cell imaging experiment illustrating the recruitment of transiently expressed eGFP-ORP1L to lysosomes following 250 µM LLOMe treatment. Cells were pre-treated for 30 min with 75 nM Lysotracker Deep Red to monitor lysosome damage (judged by the decreased number of Lysotracker spots after LLOMe treatment). The graph represents quantification of eGFP-ORP1L foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >25 cells were analyzed per experiment.


A Movie stills from a live - imaging experiment showing rapid 2xSidM - eGFP recruitment to damaged lysosomes upon 250 µM LLOMe treatment ( indicated in green ) . Lysosomes were visualized using transiently overexpressed LAMP1 - mCherry ( indicated in red ) . Yellow spots show colocalized PtdIns4P probe ( 2xSidM - eGFP ) LAMP1 - mCherry after 3 10 min of LLOMe treatment . graph shows of 2xSidM - eGFP foci per after LLOMe treatment . Error bars denote + / - from n = 3 independent live - imaging experiments , > 20 were analyzed per experiment .

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A Movie stills from a live-cell imaging experiment showing rapid 2xSidM-eGFP recruitment to damaged lysosomes upon 250 µM LLOMe treatment (indicated in green). Lysosomes were visualized using transiently overexpressed LAMP1-mCherry (indicated in red). Yellow spots show colocalized PtdIns4P probe (2xSidM-eGFP) and LAMP1-mCherry after 3 and 10 min of LLOMe treatment. The graph shows the quantification of 2xSidM-eGFP foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >20 cells were analyzed per experiment.


B HeLa were transfected with control siRNA or siRNA targeting PI4K2A or PI4K2B for 48 h transiently transfected with 2xSidM - eGFP probe to monitor PtdIns4P recruitment after LLOMe - treatment by live - imaging . Representative movie stills show dynamics of 2xSidM - eGFP recruitment in control , PI4K2A PI4K2B depleted . graph represents of 2xSidM - eGFP foci per after LLOMe - treatment . Error bars denote + / - from n = 3 independent live - imaging experiments , > 25 were analyzed per experiment for each condition .

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B HeLa cells were transfected with control siRNA or siRNA targeting PI4K2A or PI4K2B for 48 h and transiently transfected with the 2xSidM-eGFP probe to monitor PtdIns4P recruitment after LLOMe-treatment by live-cell imaging. Representative movie stills show the dynamics of 2xSidM-eGFP recruitment in control, PI4K2A and PI4K2B depleted cells. The graph represents the quantification of 2xSidM-eGFP foci per cell after LLOMe-treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >25 cells were analyzed per experiment for each condition.


C CRISPR - Cas9 mediated knockout of PI4K2A abolishes recruitment of 2xSidM - eGFP probe ( PtdIns4P ) to damaged lysosomes . Two different clones ( clone 2 clone 3 ) were tested . graph represents of 2xSidM - eGFP foci per after LLOMe - treatment . Error bars denote + / - SD from n = 2 independent live - imaging experiments , > 20 were analyzed per experiment for each condition .

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C CRISPR-Cas9 mediated knockout of PI4K2A abolishes recruitment of the 2xSidM-eGFP probe (PtdIns4P) to damaged lysosomes. Two different clones (clone 2 and clone 3) were tested. The graph represents the quantification of 2xSidM-eGFP foci per cell after LLOMe-treatment. Error bars denote +/- SD from n=2 independent live-cell imaging experiments, >20 cells were analyzed per experiment for each condition.


D Representative movie stills from live microscopy showing reduced eGFP - ORP1L recruitment in PI4K2A knockout compared to wild type HeLa upon treatment with 250 µM LLOMe . graph represents of eGFP - ORP1L foci per after LLOMe treatment . Error bars denote + / - from n = 4 independent live - imaging experiments , > 80 were analyzed per experiment for each condition .

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D Representative movie stills from live fluorescence microscopy showing reduced eGFP-ORP1L recruitment in PI4K2A knockout cells compared to wild type HeLa cells upon treatment with 250 µM LLOMe. The graph represents the quantification of eGFP-ORP1L foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=4 independent live-cell imaging experiments, >80 cells were analyzed per experiment for each condition.


Representative movie stills from live - imaging experiments monitoring Lysotracker recovery after induced lysosomal damage using 250 µM LLOMe . were pre - treated with 75 nM Lysotracker Deep Red for 30 min before addition of LLOMe . graph represents of bright Lysotracker positive foci per . While decrease in number of Lysotracker spots is quickly recovering in HeLa , there is a severe impairment in lysosomal repair in HeLa - PI4K2A - KO .

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Representative movie stills from live-cell imaging experiments monitoring Lysotracker recovery after induced lysosomal damage using 250 µM LLOMe. Cells were pre-treated with 75 nM Lysotracker Deep Red for 30 min before addition of LLOMe. The graph represents the quantification of bright Lysotracker positive foci per cell. While the decrease in the number of Lysotracker spots is quickly recovering in HeLa cells, there is a severe impairment in lysosomal repair in HeLa-PI4K2A-KO cells.


A HeLa were co - transfected with siRNAs targeting ESCRT TSG101 ALIX ( siTSG101 + siALIX ) or with non - targeting siRNA control ( siControl ) , incubated for 24 h before transfection with PtdIns4P binding probe 2xSidM - eGFP . 24 h post - transfection lysosomes were damaged with 250 µM LLOMe PtdIns4P recruitment to damaged lysosomes was monitored using 2xSidM - eGFP probe in live - imaging experiments . graph shows of 2xSidM - eGFP foci per in co - depleted of TSG101 + ALIX control ( siControl ) .

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A HeLa cells were co-transfected with siRNAs targeting ESCRT proteins TSG101 and ALIX (siTSG101+siALIX) or with non-targeting siRNA control (siControl), and incubated for 24 h before transfection with the PtdIns4P binding probe 2xSidM-eGFP. 24 h post-transfection lysosomes were damaged with 250 µM LLOMe and PtdIns4P recruitment to damaged lysosomes was monitored using the 2xSidM-eGFP probe in live-cell imaging experiments. The graph shows quantification of 2xSidM-eGFP foci per cell in cells co-depleted of TSG101+ALIX and control (siControl) cells.


A Representative movie stills of a live - imaging experiment illustrate faster recruitment of PtdIns4P probe 2xSidM - eGFP to damaged lysosomes when compared to cholesterol as indicated by mCherry - D4 probe . graph shows of 2xSidM - eGFP mCherry - D4 foci per . Error bars denote + / - SD from n = 2 independent live - imaging

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A Representative movie stills of a live-cell imaging experiment illustrate faster recruitment of the PtdIns4P probe 2xSidM-eGFP to damaged lysosomes when compared to cholesterol as indicated by the mCherry-D4 probe. The graph shows quantification of 2xSidM-eGFP and mCherry-D4 foci per cell. Error bars denote +/- SD from n=2 independent live-cell imaging experiments


D HeLa expressing an eGFP - tagged ORP1L mutant incapable of binding to VAP ( mFFAT ) show no accumulation of cholesterol reporter mCherry - D4 upon lysosomal damage induced with 250 µM LLOMe when compared to wildtype ( wt ) eGFP - ORP1L . graph shows D4 - mCherry foci per . Error bars denote + / - from n = 4 independent live - imaging experiments , > 50 were analyzed per experiment for each condition .

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D HeLa cells expressing an eGFP-tagged ORP1L mutant incapable of binding to VAP (mFFAT) show no accumulation of the cholesterol reporter mCherry-D4 upon lysosomal damage induced with 250 µM LLOMe when compared to wildtype (wt) eGFP-ORP1L. The quantification graph shows D4-mCherry foci per cell. Error bars denote +/- SEM from n=4 independent live-cell imaging experiments, >50 cells were analyzed per experiment for each condition.


A Representative movie stills from live - imaging experiments indicate recruitment of CHMP4B - eGFP to damaged lysosomes in control U18666A treated . HeLa stably expressing CHMP4B - eGFP were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe . graph shows of CHMP4B - eGFP foci per . Error bars denote + / - from n = 4 , > 30 per experiment for each condition .

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A Representative movie stills from live-cell imaging experiments indicate recruitment of CHMP4B-eGFP to damaged lysosomes in control and U18666A treated cells. HeLa cells stably expressing CHMP4B-eGFP were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe. The graph shows quantification of CHMP4B-eGFP foci per cell. Error bars denote +/- SEM from n=4, >30 cells per experiment for each condition.


B Representative immunofluorescence illustrate Galectin - 3 recruitment kinetics on damaged lysosomes upon treatment with 2 µM U18666A for 17 h . HeLa were treated or not ( Control ) with U18666A + / - LLOMe for indicated time points , fixed processed for immunofluorescence microscopy . Endogenous of Galectin - 3 were visualized .

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B Representative immunofluorescence images illustrate Galectin-3 recruitment kinetics on the damaged lysosomes upon treatment with 2 µM U18666A for 17 h. HeLa cells were treated or not (Control) with U18666A and +/- LLOMe for the indicated time points, fixed and processed for immunofluorescence microscopy. Endogenous levels of Galectin-3 were visualized.


C Number of Galectin - 3 positive dots per was automatically quantified from dataset described in B . Error bars denote + / - from n = 3 independent experiments . > 225 were analyzed per condition , distribution visualized as violin plots .

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C Number of Galectin-3 positive dots per cell was automatically quantified from the dataset described in B. Error bars denote +/- SEM from n=3 independent experiments. >225 cells were analyzed per condition, distribution visualized as violin plots.


D Representative movie stills from live - imaging experiments indicate recruitment of PtdIns4P probe 2xSidM - eGFP to damaged lysosomes in control but not in U18666A treated . HeLa were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe . graph shows of 2xSidM - eGFP foci per .

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D Representative movie stills from live-cell imaging experiments indicate recruitment of the PtdIns4P probe 2xSidM-eGFP to damaged lysosomes in control but not in the U18666A treated cells. HeLa cells were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe. The graph shows quantification of 2xSidM-eGFP foci per cell.


A Representative movie stills of a live - imaging experiment show recruitment dynamics of transiently expressed eGFP - OSBP in HeLa treated with 250 µM LLOMe . graph shows quantifications of OSBP foci per . Error bars denote + / - from n = 5 independent live - imaging experiments , > 22 per experiment .

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A Representative movie stills of a live-cell imaging experiment show recruitment dynamics of transiently expressed eGFP-OSBP in HeLa cells treated with 250 µM LLOMe. The graph shows quantifications of OSBP foci per cell. Error bars denote +/- SEM from n=5 independent live-cell imaging experiments, >22 cells per experiment.


D Knock down efficiency of siRNA depletion of OSBP in HeLa as detected by Western blot . β - actin used as a loading control .

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D Knock down efficiency of siRNA depletion of OSBP in HeLa cells as detected by Western blot. β-actin used as a loading control.