Figure 5A ) CrizR1 cells were treated with vehicle control , 200nM alvocidib or 100nM THZ1 for 6 hrs , then chromatin was precipitated with an anti - RNA polymerase II antibody and sequenced . Plotted is the average number of peaks per condition . GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS ( ± 1kb ) with ( B ) alvocidib Shown on the right is the plot of the " MYC targets " - enriched signature . GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS ( ± 1kb ) with ( C ) THZ1 treatment . Shown on the right is the plot of the " MYC targets " - enriched signature . D ) Gene tracks of RNA polymerase II occupancy at the MCL1 , BIRC5 ( Survivin ) , CCND1 and MYC genes . E ) qPCR of MYC and CCND1 upon treatment of CrizR1 cells with Alvocidib or THZ1 for 6h . The graph represents the mean fold change ± SD . MYC : alvocidib vs DMSO P = 0 . 03 , THZ1 vs DMSO P < 0 . 0001 ; CCND1 : alvocidib vs DMSO P = 0 . 001 , THZ1 vs DMSO P = 0 . 003 . F ) BIM is upregulated upon MYC silencing in CrizR1 cells . G ) Annexin V assay showing the percentage of apoptotic CrizR1 cells upon MYC downregulation using siRNA . Early apoptosis P = 0 . 002 , late apoptosis P = 0 . 04 , alive P = 0 . 0008 . H ) Annexin V assay showing the percentage of apoptotic CrizR1 cells after treatment with trametinib , rapamycin or both . Trametinib : early apoptosis P = 0 . 002 , late apoptosis P = 0 . 05 , alive P = 0 . 009 ; rapamycin : early apoptosis P = 0 . 04 , late apoptosis P = 0 . 8 , alive P = 0 . 003 ; tram + rapa : early apoptosis P = 0 . 0001 , late apoptosis P = 0 . 04 , alive P = 0 . 01 .

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Figure 5A) CrizR1 cells were treated with vehicle control, 200nM alvocidib or 100nM THZ1 for 6 hrs, then chromatin was precipitated with an anti-RNA polymerase II antibody and sequenced. Plotted is the average number of peaks per condition.GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (B) alvocidib Shown on the right is the plot of the "MYC targets"-enriched signature.GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (C) THZ1 treatment. Shown on the right is the plot of the "MYC targets"-enriched signature.D) Gene tracks of RNA polymerase II occupancy at the MCL1, BIRC5 (Survivin), CCND1 and MYC genes.E) qPCR of MYC and CCND1 upon treatment of CrizR1 cells with Alvocidib or THZ1 for 6h. The graph represents the mean fold change ± SD. MYC: alvocidib vs DMSO P =0.03, THZ1 vs DMSO P <0.0001; CCND1: alvocidib vs DMSO P =0.001, THZ1 vs DMSO P =0.003.F) BIM is upregulated upon MYC silencing in CrizR1 cells.G) Annexin V assay showing the percentage of apoptotic CrizR1 cells upon MYC downregulation using siRNA. Early apoptosis P =0.002, late apoptosis P=0.04, alive P =0.0008.H) Annexin V assay showing the percentage of apoptotic CrizR1 cells after treatment with trametinib, rapamycin or both. Trametinib: early apoptosis P =0.002, late apoptosis P =0.05, alive P =0.009; rapamycin: early apoptosis P =0.04, late apoptosis P =0.8, alive P =0.003; tram+rapa: early apoptosis P =0.0001, late apoptosis P =0.04, alive P =0.01.


Figure 6Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to crizotinib and correspondent tumour weights ( P . O . control n = 5 ; I . P . control n = 4 ; crizotinib n = 8 Tumour weights , crizotinib P = 0 . 44Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to alvocidib and correspondent tumour weights ( P . O . control n = 5 ; I . P . control n = 4 and alvocidib n = 6 ) . Tumour weights , , alvocidib P = 0 . 03 . C ) Tumour growth of in vivo xenografts of Ste - 1 AlectR cell lines in response to alvocidib and correspondent tumor weights ( I . P . control n = 6 ; alvocidib n = 4 ) . Tumour weights alvocidib P = 0 . 004 . D ) Number of cleaved caspase - 3 positive nuclei in alvocidib treated tumours compared to controls . E ) Ki67 and p21 staining of CrizR1 xenograft tumours . Scale bar = 200μm . Quantitative analysis of IHC staining is presented on the right . Ki67 P = 0 . 0004 ; p21 P = 0 . 02 . F ) qPCR of Survivin ( P = 0 . 0009 ) , MCL - 1 ( P = 0 . 04 ) , MYC ( P = 0 . 007 ) and BIM ( P = 0 . 03 ) in Ste - 1 AlectR cells xenografts treated with alvocidib ( Control n = 6 ; alvocidib n = 4 ) . G ) Proliferation assay of H3122 parental and isogenic drug - resistant cell lines , treated with DMSO , 100nM lorlatinib , 200nM alvocidib or 50nM dinaciclib for 72h ( n = 4 ) . H ) Growth curve of CrizR1 xenograft tumors in response to lorlatinib ( Control n = 7 ; lorlatinib n = 7 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to crizotinib and correspondent tumour weights (P.O. control n=5; I.P. control n=4; crizotinib n=8 Tumour weights, crizotinib P= 0.44Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to alvocidib and correspondent tumour weights (P.O. control n=5; I.P. control n=4 and alvocidib n=6). Tumour weights, , alvocidib P=0.03.C) Tumour growth of in vivo xenografts of Ste-1 AlectR cell lines in response to alvocidib and correspondent tumor weights (I.P. control n=6; alvocidib n=4). Tumour weights alvocidib P=0.004.D) Number of cleaved caspase-3 positive nuclei in alvocidib treated tumours compared to controls.E) Ki67 and p21 staining of CrizR1 xenograft tumours. Scale bar = 200μm. Quantitative analysis of IHC staining is presented on the right. Ki67 P=0.0004; p21 P=0.02.F) qPCR of Survivin (P=0.0009), MCL-1 (P=0.04), MYC (P=0.007) and BIM (P=0.03) in Ste-1 AlectR cells xenografts treated with alvocidib (Control n=6; alvocidib n=4).G) Proliferation assay of H3122 parental and isogenic drug-resistant cell lines, treated with DMSO, 100nM lorlatinib, 200nM alvocidib or 50nM dinaciclib for 72h (n=4).H) Growth curve of CrizR1 xenograft tumors in response to lorlatinib (Control n=7; lorlatinib n=7).


Figure 1Immunoblot analysis of indicated cell lines , starved for 6 hours ( HBSS ) as indicated , and treated with 25 μM MG132 or 200 ng / ml of bafilomycin A1 ( Baf A1 ) for the indicated times . In C endogenous CALCOCO1 is analysed and the bars represent the mean ± sd of band intensities relative to the actin loading control , as quantified using ImageJ of three independent experiments . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . Immunoblot analysis of indicated cell lines , starved for 6 hours ( HBSS ) as indicated , and treated with 25 μM MG132 or 200 ng / ml of bafilomycin A1 ( Baf A1 ) for the indicated times . In E , endogenous CALCOCO1 is analysed and the bars represent the mean ± sd of band intensities relative to the actin loading control , as quantified using ImageJ of three independent experiments . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . F A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 and immunostained for endogenous GM130 . Scale bars are 5 μm and 2 μm ( zoomed inset ) . G Same cells as in E were left untreated or treated with Baf A1 for 6 hours , and then immunostained for endogenous p62 and LC3B . Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans . H CALCOCO1 , p62 and LC3B puncta in in the indicated conditions , counted using an automated system . The error bars represent mean ± SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test . Significance is displayed as * * * p < 0 . 001 . I Percentage of co - localization of CALCOCO1 puncta with p62 and / or LC3B in cells treated as indicated . The error bars represent mean ± SEM of three independent experiments per condition and over 250 cells per experiment . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test . Significance is displayed as * * * p < 0 . 001 . J HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1 . Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times.In C endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In E, endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.F A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 and immunostained for endogenous GM130. Scale bars are 5 μm and 2 μm (zoomed inset).G Same cells as in E were left untreated or treated with Baf A1 for 6 hours, and then immunostained for endogenous p62 and LC3B. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.H CALCOCO1, p62 and LC3B puncta in in the indicated conditions, counted using an automated system. The error bars represent mean±SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001.I Percentage of co-localization of CALCOCO1 puncta with p62 and/or LC3B in cells treated as indicated. The error bars represent mean±SEM of three independent experiments per condition and over 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001.J HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.


Figure 2A GST pulldown binding assay of in vitro transcribed / translated 35S - Myc - CALCOCO1 with recombinant GST - tagged ATG8 family proteins . GST and GST fusion proteins were visualized by coomassie brilliant blue staining ( bottom panel ) , and the co - precipitated Myc - CALCOCO1 was detected by autoradiography ( upper panel ) . The numbers below the AR represent % binding in the shown AR . B GST pulldown assay of transiently transfected Myc - CALCOCO1 from HEK293 cell extracts with recombinant GST - tagged ATG8 family proteins . GST and GST fusions were visualized by Ponceau S staining ( bottom panel ) , and co - precipitated Myc - CALCOCO1 detected by immunoblotting with anti - Myc antibody ( upper panel ) . GST pulldown assays of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteins . Precipitated GST and GST fusions and co - precipitated Myc - CALCOCO1 constructs were analyzed as in A . GST pulldown assays of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteins . Precipitated GST and GST fusions and co - precipitated Myc - CALCOCO1 constructs were analyzed as in A . GST pulldown assays of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteins . Precipitated GST and GST fusions and co - precipitated Myc - CALCOCO1 constructs were analyzed as in A . GST pulldown assays of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteins . Precipitated GST and GST fusions and co - precipitated Myc - CALCOCO1 constructs were analyzed as in A . GST pulldown assays of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteins . Precipitated GST and GST fusions and co - precipitated Myc - CALCOCO1 constructs were analyzed as in A . I GST pulldown assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST - tagged CALCOCO1 constructs . GST and GST fusions were visualized as in A , and co - precipitated GABARAP with anti - GABARAP antibody ( upper panel ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A GST pulldown binding assay of in vitro transcribed/translated 35S-Myc-CALCOCO1 with recombinant GST-tagged ATG8 family proteins. GST and GST fusion proteins were visualized by coomassie brilliant blue staining (bottom panel), and the co-precipitated Myc-CALCOCO1 was detected by autoradiography (upper panel). The numbers below the AR represent % binding in the shown AR .B GST pulldown assay of transiently transfected Myc-CALCOCO1 from HEK293 cell extracts with recombinant GST-tagged ATG8 family proteins. GST and GST fusions were visualized by Ponceau S staining (bottom panel), and co-precipitated Myc-CALCOCO1 detected by immunoblotting with anti-Myc antibody (upper panel).GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.I GST pulldown assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST-tagged CALCOCO1 constructs. GST and GST fusions were visualized as in A, and co-precipitated GABARAP with anti-GABARAP antibody (upper panel).


Figure 3GST pulldowns testing binding of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteins ( left ) . Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs . The presence of two well separated binding surfaces on ATG8 proteins binding to LIR ( LDS ) and UIR ( UDS ) is indicated ( right ) . B GST pulldowns testing binding of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteinsC GST pulldown assays of in vitro transcribed / translated 35S - Myc - CALCOCO1 and 35S - Myc - p62 with recombinant GST - GABARAPL2 ( WT and indicated mutants ) . D GST pulldown assays of in vitro transcribed / translated 35S - Myc - TAX1BP1 ( WT and indicated mutants ) with recombinant GST - GABARAP ( WT and indicated mutants ) . Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP - CALCOCO1 or EGFP - CALCOCO1 mLIR + ∆ 623 - 691 . Cells were induced with tetracycline for 24 hours and then starved or treated with MG132 or Baf A1 as indicated . The blot panels are from more than one western blot experiment but for clarity , only a single actin / GAPDH loading control is shown . In F , the bars represent the mean ± sd of band intensities relative to the actin or GAPDH loading controls of three independent experiments 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 . 01 ; ns is not significant . G HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 or EGFP - CALCOCO1 mLIR + ∆ 623 - 691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B . Scale bars , 5 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins (left). Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs. The presence of two well separated binding surfaces on ATG8 proteins binding to LIR (LDS) and UIR (UDS) is indicated (right).B GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteinsC GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 and 35S-Myc-p62 with recombinant GST-GABARAPL2 (WT and indicated mutants).D GST pulldown assays of in vitro transcribed/translated 35S-Myc-TAX1BP1 (WT and indicated mutants) with recombinant GST-GABARAP (WT and indicated mutants).Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691. Cells were induced with tetracycline for 24 hours and then starved or treated with MG132 or Baf A1 as indicated. The blot panels are from more than one western blot experiment but for clarity, only a single actin/GAPDH loading control is shown. In F, the bars represent the mean±sd of band intensities relative to the actin or GAPDH loading controls of three independent experiments 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 ˂ 0.01; ns is not significant.G HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B. Scale bars, 5 μm.


Figure 4HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 were starved for 4 hours and then immunostained with anti - ATG13 and anti - WIPI2 antibodies . Scale bars in A are 5 μm for the confocal microscopy images and 2 μm for the airyscans . In B , the error bars represent mean ± sd of puncta per cell from three independent experiments and100 - 200 cells per each experiment . Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH In D the bars represent the mean ± sd of band intensities relative to the GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH In , the bars represent the mean ± sd of band intensities relative to the GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin ) . In , the bars represent the mean ± sd of band intensities relative to the actin loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . , I the bars represent the mean ± sd of band intensities relative to the actin loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDHImmunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH The asterisk in K indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP - CALCOCO1 in cells extracts from the rescued cells . In the bars represent the mean ± sd of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were starved for 4 hours and then immunostained with anti-ATG13 and anti-WIPI2 antibodies. Scale bars in A are 5 μm for the confocal microscopy images and 2 μm for the airyscans.In B, the error bars represent mean±sd of puncta per cell from three independent experiments and100-200 cells per each experiment.Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH In D the bars represent the mean±sd of band intensities relative to the GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH In , the bars represent the mean±sd of band intensities relative to the GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin). In , the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant., I the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDHImmunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH The asterisk in K indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP-CALCOCO1 in cells extracts from the rescued cells. In the bars represent the mean±sd of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Figure 5B Co - IP of Myc - VAPA or Myc - VAPB with EGFP - CALCOCO1 from transiently transfected HEK293 cells . The asterisks ( * ) indicate Myc - VAP previously detected on the same membraneC , D GST pulldown assays of in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - VAPA or GST - VAPB constructs . The scheme is a representation of CALCOCO1 domains and motifs . E HeLa cells transiently co - transfected with EGFP - CALCOCO1 and Myc - VAPA or - VAPB were immunostained with anti - Myc antibody . Scale bars , 5 μm .

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Figure 5B Co- IP of Myc-VAPA or Myc-VAPB with EGFP-CALCOCO1 from transiently transfected HEK293 cells. The asterisks (*) indicate Myc-VAP previously detected on the same membraneC, D GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-VAPA or GST-VAPB constructs.The scheme is a representation of CALCOCO1 domains and motifs.E HeLa cells transiently co-transfected with EGFP-CALCOCO1 and Myc-VAPA or -VAPB were immunostained with anti-Myc antibody. Scale bars, 5 μm.


Figure 6A , 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 analysis of HeLa cells transfected with the indicated siRNAs . 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 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 .

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Figure 6A, 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 analysis of HeLa cells transfected with the indicated siRNAs.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 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.


Figure 7A , 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 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 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 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 analysis of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 in fed or starved conditions and transfected with the indicated VAPA / B siRNAs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A, 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 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 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 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 analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 in fed or starved conditions and transfected with the indicated VAPA/B siRNAs.


Figure 8A 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 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 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 test and significance displayed as * * * p ˂ 0 . 001 ; ns is not significant , n = 3 .

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Figure 8A 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 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 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 test and significance displayed as ***p ˂ 0.001; ns is not significant, n=3.


Figure 2A 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 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 - 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A 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 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-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.


Figure 3A 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 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 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 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 .

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Figure 3A 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 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 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 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.


Figure 4Representative 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 .

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Figure 4Representative 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.


Figure 5A 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 .

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Figure 5A 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.


Figure 6A 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 experimentsD 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 .

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Figure 6A 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 experimentsD 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.


Figure 7A 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 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 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 - 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 .

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Figure 7A 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 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 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-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.


Figure 8A 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 cells as detected by Western blot . β - actin used as a loading control .

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Figure 8A 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 cells as detected by Western blot. β-actin used as a loading control.