( P ) The effect of overexpression of consitutively active CDC42 on ABCA1 abundance in MIN6 cells with ABCA12 - deficiency . Treatments are defined in the table below .

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(P) The effect of overexpression of consitutively active CDC42 on ABCA1 abundance in MIN6 cells with ABCA12-deficiency. Treatments are defined in the table below.


( Q ) Effects of Jasplakinolide on F - actin levels ( green ) in siRNA treated cells ( scale bar = 10μm ) .

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(Q) Effects of Jasplakinolide on F-actin levels (green) in siRNA treated cells (scale bar=10μm).


( R ) Effects of Abca12 knockdown and Jasplakinolide treatment on MIN6 cell in low and high glucose ( mean ± SEM , n = 4 biological replicates , * p = < 0 . 05 , * * p = < 0 . 01 , significance relative to untreated cells , Students t - test )

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(R) Effects of Abca12 knockdown and Jasplakinolide treatment on MIN6 cell in low and high glucose (mean±SEM, n=4 biological replicates, *p=<0.05, **p=<0.01, significance relative to untreated cells, Students t-test)


( A ) Mass spectroscopy analysis of levels of cholesterol and cholesteryl esters in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age ( mean ± SEM , n = 10 , 4 and 5 mice per genotype respectively ; COH = cholesterol , CE = cholesteryl ester ) .

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(A) Mass spectroscopy analysis of levels of cholesterol and cholesteryl esters in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively; COH=cholesterol, CE=cholesteryl ester).


( B ) Mass spectroscopy analysis of lipids families in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age ( mean ± SEM , n = 10 , 4 and 5 mice per genotype respectively , * * p = < 0 . 01 Students t - test ) ; CE = cholesteryl ester , Cer = ceramide , DHC ( Hex2Cer ) = dihexosylceramide , MHC ( HexCer ) = monohexosylceramide , SM = sphingomyelin , GM3 = GM3 ganglioside , dhCer = dihydroceramide , DG = diacylglyerol , TG = triacylglycerol ) .

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(B) Mass spectroscopy analysis of lipids families in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively, **p=<0.01 Students t-test); CE=cholesteryl ester, Cer=ceramide, DHC (Hex2Cer)=dihexosylceramide, MHC (HexCer)=monohexosylceramide, SM=sphingomyelin, GM3=GM3 ganglioside, dhCer=dihydroceramide, DG=diacylglyerol, TG=triacylglycerol).


( C ) Mass spectroscopy analysis of triglyceride species in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age ( mean ± SEM , n = 10 , 4 and 5 mice per genotype respectively , * p = < 0 . 05 , * * p = < 0 . 01 , Students t - test )

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(C) Mass spectroscopy analysis of triglyceride species in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively, *p=<0.05, **p=<0.01, Students t-test)


( D ) Immunohistochemical staining of levels IL - 1β expression in sections of pancreas from Abca12tm1d mice versus control animals at 24 weeks of age ( bar = 25μm , islets outlined ) .

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(D) Immunohistochemical staining of levels IL-1β expression in sections of pancreas from Abca12tm1d mice versus control animals at 24 weeks of age (bar = 25μm, islets outlined).


( E ) Quantification of islet specific IL1β staining from sections of Abca12tm1d , Cre and wild type mice ( mean ± SEM , n = 3 , 3 and 4 mice of each genotype respectively ; * p = < 0 . 05 , Students t - test ) .

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(E) Quantification of islet specific IL1β staining from sections of Abca12tm1d, Cre and wild type mice (mean±SEM, n=3, 3 and 4 mice of each genotype respectively; *p=<0.05, Students t-test).


Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing β - cell mass ( F ) ( I ) ( mean ± SEM , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - test ) .

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Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing β-cell mass (F) (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).


Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing cleaved caspase 3 ( G ) ( I ) ( mean ± SEM , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - test ) .

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Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing cleaved caspase 3 (G) (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).


Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing glucagon ( H ) ( I ) ( mean ± SEM , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - test ) .

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Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing glucagon (H) (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).


Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing F480 + macrophage populations ( I ) ( mean ± SEM , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - test ) .

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Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing F480+ macrophage populations (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).


( J ) Fasting blood glucose levels coincident with the emergence of inflammatory phenotypes ( mean ± SEM , n = 3 , 4 and 5 mice per genotype respectively , * p = < 0 . 05 , Students t - test ) .

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(J) Fasting blood glucose levels coincident with the emergence of inflammatory phenotypes (mean±SEM, n=3, 4 and 5 mice per genotype respectively, *p=<0.05, Students t-test).


B ) Proliferation assay of H3122 parental and isogenic drug - resistant cells , treated with the indicated concentrations of crizotinib for 72h . P < 0 . 0001 was calculated for IC50 shift as indicated in the methods .

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B) Proliferation assay of H3122 parental and isogenic drug-resistant cells, treated with the indicated concentrations of crizotinib for 72h. P < 0.0001 was calculated for IC50 shift as indicated in the methods.


C ) Gene - set enrichment analysis after RNA - seq of H3122 sensitive vs CrizR1 crizotinib resistant cells . D ) Epithelial to mesenchymal transition enrichment plot from the RNA - seq of C ) , using the HALLMARK gene collection .

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C) Gene-set enrichment analysis after RNA-seq of H3122 sensitive vs CrizR1 crizotinib resistant cells. D) Epithelial to mesenchymal transition enrichment plot from the RNA-seq of C), using the HALLMARK gene collection.


E ) H3122 parental and drug - resistant isogenic cell lines were RNA - extracted and gene expression was analysed by RT - qPCR . On the right hand side is a western blot analysis of the AXL and EML4 - ALK protein levels from the same cell lines . AXL : CrizR1 vs H3122 P < 0 . 001 , CrizR4 vs H3122 P < 0 . 001 , CrizR5 vs H3122 P = 0 . 03 , CeritR vs H3122 P = 0 . 2 ; LOX : CrizR1 vs H3122 P < 0 . 001 , CrizR4 vs H3122 P < 0 . 001 , CrizR5 vs H3122 P < 0 . 001 , CeritR vs H3122 P = 0 . 008 ; SNAI2 : CrizR1 vs H3122 P < 0 . 001 , CrizR4 vs H3122 P < 0 . 001 , CrizR5 vs H3122 P = 0 . 003 , CeritR vs H3122 P = 0 . 003 ; VIM : CrizR1 vs H3122 P < 0 . 001 , CrizR4 vs H3122 P < 0 . 001 , CrizR5 vs H3122 P < 0 . 001 , CeritR vs H3122 P < 0 . 001 .

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E) H3122 parental and drug-resistant isogenic cell lines were RNA-extracted and gene expression was analysed by RT-qPCR. On the right hand side is a western blot analysis of the AXL and EML4-ALK protein levels from the same cell lines.AXL: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P=0.03, CeritR vs H3122 P=0.2; LOX: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P <0.001, CeritR vs H3122 P=0.008; SNAI2: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P =0.003, CeritR vs H3122 P =0.003; VIM: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P <0.001, CeritR vs H3122 P <0.001.


F ) Gene expression analysis by RT - qPCR of CrizR1 cells treated with the indicated concentrations of bemcentinib for 48h . LOX : P = 0 . 03 ; SNAI2 : P = 0 . 02 ; VIM : P = 0 . 03 .

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F) Gene expression analysis by RT-qPCR of CrizR1 cells treated with the indicated concentrations of bemcentinib for 48h. LOX: P=0.03; SNAI2: P =0.02; VIM: P =0.03.


G ) Proliferation assay of CrizR1 and CrizR4 cells treated with the indicated concentrations of bemcentinib ± 1uM crizotinib for 72h . * * * = P < 0 . 001 for IC50 shift , as indicated in the methods ( n = 4 ) .

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G) Proliferation assay of CrizR1 and CrizR4 cells treated with the indicated concentrations of bemcentinib ± 1uM crizotinib for 72h. *** = P < 0.001 for IC50 shift, as indicated in the methods (n=4).


H ) Annexin V + apoptotic assay in CrizR1 cells treated with 1uM crizotinib , 2 . 5uM bemcentinib , or combination . STE - 1 parental cells were used as crizotinib drug control . crizotinib : P = 0 . 2 ; bemcentinib : P = 0 . 4 ; combination : P = 0 . 3 .

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H) Annexin V+ apoptotic assay in CrizR1 cells treated with 1uM crizotinib, 2.5uM bemcentinib, or combination. STE-1 parental cells were used as crizotinib drug control. crizotinib: P =0.2; bemcentinib: P =0.4; combination: P=0.3.


A ) GSEA enrichment analysis using the KEGG gene set identifiers . Shown are the significantly dysregulated pathways ( P < 0 . 05 ) . B ) Cell cycle enrichment plot from A ) .

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A) GSEA enrichment analysis using the KEGG gene set identifiers. Shown are the significantly dysregulated pathways (P < 0.05). B) Cell cycle enrichment plot from A).


C ) Western blot analysis of H3122 parental and isogenic drug - resistant cell lines for the indicated proteins .

C) Western blot analysis of H3122 parental and isogenic drug-resistant cell lines for the indicated proteins.


D ) Western blot analysis of Ste - 1 parental and isogenic Alectinib resistant cell lines for the indicated proteins .

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D) Western blot analysis of Ste-1 parental and isogenic Alectinib resistant cell lines for the indicated proteins.


E ) Proliferation assay of CrizR1 cells treated with the indicated concentrations of alvocidib ± 1uM crizotinib for 72h . P = 0 . 2 ( n = 4 ) .

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E) Proliferation assay of CrizR1 cells treated with the indicated concentrations of alvocidib ± 1uM crizotinib for 72h. P=0.2 (n=4).


F ) As above , in a proliferation assay , H3122 and CrizR1 cells were treated in parallel with DMSO or 1uM crizotinib as drug control . H3122 vs DMSO P < 0 . 0001 , CrizR1 vs DMSO P = 0 . 1 ( n = 4 ) .

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F) As above, in a proliferation assay, H3122 and CrizR1 cells were treated in parallel with DMSO or 1uM crizotinib as drug control. H3122 vs DMSO P <0.0001, CrizR1 vs DMSO P=0.1 (n=4).


Proliferation assay of CrizR1 , CrizR4 , CrizR5 , CeritR isogenic drug - resistant cell lines , treated with the indicated concentrations of alvocidib ( G ) for 72h . P > 0 . 05 was calculated for IC50 shift , as indicated in the methods .

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Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR isogenic drug-resistant cell lines, treated with the indicated concentrations of alvocidib(G) for 72h. P >0.05 was calculated for IC50 shift, as indicated in the methods.


H ) Proliferation assay of AlecR isogenic drug - resistant cell lines , treated with the indicated concentrations of dinaciclib ( H ) for 72h . P > 0 . 05 was calculated for IC50 shift , as indicated in the methods .

H) Proliferation assay of AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of dinaciclib (H) for 72h. P >0.05 was calculated for IC50 shift, as indicated in the methods.


A ) Proliferation assay of CrizR1 , CrizR4 , CrizR5 , CeritR , and AlecR isogenic drug - resistant cell lines , treated with the indicated concentrations of THZ1 for 72h . P < 0 . 05 was calculated for IC50 shift , as indicated in the methods .

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A) Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR, and AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of THZ1 for 72h. P <0.05 was calculated for IC50 shift, as indicated in the methods.


B ) Crystal violet staining of EML4 / ALK parental and drug - resistant cells . Cells were treated with the indicated drugs until the vehicle control reached confluence , then fixed and stained .

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B) Crystal violet staining of EML4/ALK parental and drug-resistant cells. Cells were treated with the indicated drugs until the vehicle control reached confluence, then fixed and stained.


C ) CrizR1 cells were treated with the indicated drugs for 72h , then cells were stained with Annexin V / PI . Flow cytometry was then used to quantify Annexin V + cells . Alvocidib : early apoptosis P = 0 . 002 , late apoptosis P = 0 . 006 , alive P = 0 . 0008 ; Dinaciclib : early apoptosis P = 0 . 005 , late apoptosis P = 0 . 02 , alive P = 0 . 002 .

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C) CrizR1 cells were treated with the indicated drugs for 72h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells. Alvocidib: early apoptosis P=0.002, late apoptosis P=0.006, alive P=0.0008; Dinaciclib: early apoptosis P=0.005, late apoptosis P=0.02, alive P=0.002.


A ) Indicated cell lines were treated with DMSO , 200nM alvocidib or 25nM dinaciclib for 48h , then cells were stained with Annexin V / PI . Flow cytometry was then used to quantify Annexin V + cells ( n = 2 ) . Alvocidib : CrizR1 P = 0 . 005 , AlecR P = 0 . 001 , PC9 P = 0 . 05 , A549 P = 0 . 002 , H1299 P = 0 . 03 , H460 P = 0 . 07 ; P dinaciclib : CrizR1 P = 0 . 02 , AlecR P = 0 . 005 , PC9 P = 0 . 06 , A549 P = 0 . 08 , H1299 P = 0 . 09 , H460 P = 0 . 01 .

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A) Indicated cell lines were treated with DMSO, 200nM alvocidib or 25nM dinaciclib for 48h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells (n=2). Alvocidib: CrizR1 P=0.005, AlecR P =0.001, PC9 P =0.05, A549 P =0.002, H1299 P =0.03, H460 P =0.07; P dinaciclib: CrizR1 P=0.02, AlecR P =0.005, PC9 P =0.06, A549 P =0.08, H1299 P =0.09, H460 P =0.01.


( B ) CrizR1 cells were treated with DMSO or 200nM alvocidib for 24h and cell extracts were hybridized to a 43 - antibody array and analysed by immunoblotting . Graphs depict the significant changes from two independent experiments .

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(B) CrizR1 cells were treated with DMSO or 200nM alvocidib for 24h and cell extracts were hybridized to a 43-antibody array and analysed by immunoblotting. Graphs depict the significant changes from two independent experiments.


C ) CrizR1 / CeritR / AlecR cells were treated with DMSO or 200nM alvocidib or 50nM dinaciclib for 6h and cell extracts were analysed by western blotting .

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C) CrizR1/CeritR/AlecR cells were treated with DMSO or 200nM alvocidib or 50nM dinaciclib for 6h and cell extracts were analysed by western blotting.


D ) ( Top ) Western blot analysis of CrizR1 cells treated with siScrambled or siRNA for CDK7 or CDK9 for 72h . ( Bottom ) CrizR1 were treated as above , or with DMSO / alvocidib / THZ1 and cells were stained with Annexin V / PI and analysed by flow cytometry for Annexin V + cells 72h post - transfection ( n = 2 ) . Annexin siCDK7 P = 0 . 2 , siCDK9 P = 0 . 015 , alvocidib P = 0 . 0001 , THZ1 P = 0 . 004 .

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D) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or siRNA for CDK7 or CDK9 for 72h. (Bottom) CrizR1 were treated as above, or with DMSO/alvocidib/THZ1 and cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection (n=2). Annexin siCDK7 P=0.2, siCDK9 P=0.015, alvocidib P=0.0001, THZ1 P =0.004.


E ) H3122 parental and CrizR1 cells were treated with DMSO , 200nM alvocidib or 250ng / ml actinomycin D for 6 hours . RNA was extracted and the mRNA levels of the indicated genes were quantified by RT - qPCR . CDK1 : DMSO vs H3122 DMSO P = 0 . 002 , Actinomycin vs DMSO P = 0 . 0003 , Alvocidib vs DMSO P = 0 . 0005 ; CDK2 : DMSO vs H3122 DMSO P = 0 . 0001 , Actinomycin vs DMSO P < 0 . 0001 , Alvocidib vs DMSO P = 0 . 0005 ; CDK6 : DMSO vs H3122 DMSO P = 0 . 0005 , Actinomycin vs DMSO P = 0 . 0004 , Alvocidib vs DMSO P = 0 . 0004 ; CDK9 : DMSO vs H3122 DMSO P = 0 . 0003 , Actinomycin vs DMSO P = 0 . 0002 , Alvocidib vs DMSO P = 0 . 0003 ; CCNB1 : DMSO vs H3122 DMSO P = 0 . 02 , Actinomycin vs DMSO P = 0 . 0002 , Alvocidib vs DMSO P = 0 . 0003 ; CCNE1 : DMSO vs H3122 DMSO P = 0 . 0004 , Actinomycin vs DMSO P = 0 . 0004 , Alvocidib vs

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E) H3122 parental and CrizR1 cells were treated with DMSO, 200nM alvocidib or 250ng/ml actinomycin D for 6 hours. RNA was extracted and the mRNA levels of the indicated genes were quantified by RT-qPCR. CDK1: DMSO vs H3122 DMSO P=0.002, Actinomycin vs DMSO P=0.0003, Alvocidib vs DMSO P=0.0005; CDK2: DMSO vs H3122 DMSO P =0.0001, Actinomycin vs DMSO P<0.0001, Alvocidib vs DMSO P=0.0005; CDK6: DMSO vs H3122 DMSO P =0.0005, Actinomycin vs DMSO P =0.0004, Alvocidib vs DMSO P =0.0004; CDK9: DMSO vs H3122 DMSO P=0.0003, Actinomycin vs DMSO P =0.0002, Alvocidib vs DMSO P =0.0003; CCNB1: DMSO vs H3122 DMSO P=0.02, Actinomycin vs DMSO P=0.0002, Alvocidib vs DMSO P=0.0003; CCNE1: DMSO vs H3122 DMSO P =0.0004, Actinomycin vs DMSO P =0.0004, Alvocidib vs DMSO


F ) RT - qPCR analysis of MCL - 1 and Survivin expression after treatment of CrizR1 cells with 100nM alvocidib , 25nM dinaciclib and 50 nM THZ1 . RNA was extracted after 24h of treatment . MCL - 1 alvocidib vs control P = 0 . 0001 , dinaciclib vs control P = 0 . 0001 , THZ1 vs control P = 0 . 0001 ; Survivin : alvocidib vs control P = 0 . 01 , dinaciclib vs control P = 0 . 01 , THZ1 vs control P = 0 . 0001 .

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F) RT-qPCR analysis of MCL-1 and Survivin expression after treatment of CrizR1 cells with 100nM alvocidib, 25nM dinaciclib and 50 nM THZ1. RNA was extracted after 24h of treatment. MCL-1 alvocidib vs control P=0.0001, dinaciclib vs control P=0.0001, THZ1 vs control P =0.0001; Survivin: alvocidib vs control P =0.01, dinaciclib vs control P=0.01, THZ1 vs control P =0.0001.


G ) ( Top ) Western blot analysis of CrizR1 cells treated with siScrambled or with a pool of 4 different siRNAs targeting Survivin ( siBIRC5 ) . ( Bottom ) Cells were stained with Annexin V / PI and analysed by flow cytometry for Annexin V + cells 72h post - transfection . Annexin : early apoptosis P = 0 . 0003 , late apoptosis P = 0 . 5 , alive P < 0 . 0001 .

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G) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or with a pool of 4 different siRNAs targeting Survivin (siBIRC5). (Bottom) Cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection. Annexin: early apoptosis P =0.0003, late apoptosis P =0.5, alive P <0.0001.


H ) The CCLE RNA - seq dataset was used and RPKM values were plotted for the Cyclin D1 and Survivn genes , indicating high expression in H3122 cells compared with other LUAD cells .

H) The CCLE RNA-seq dataset was used and RPKM values were plotted for the Cyclin D1 and Survivn genes, indicating high expression in H3122 cells compared with other LUAD cells.


A ) 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 .

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A) 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 .

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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 .

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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 .

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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 .

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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 .

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 .

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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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.


Tumour 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 .

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Tumour 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= 0.44


Tumour 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 .

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Tumour 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 .

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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 .

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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 .

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 ) .

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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 ) .

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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 ) .

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H) Growth curve of CrizR1 xenograft tumors in response to lorlatinib (Control n=7; lorlatinib n=7).


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 .

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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 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 .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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.


A 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 .

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A 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 ) .

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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 .

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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 ) .

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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).


GST 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 ) .

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GST 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

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B GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins


C GST pulldown assays of in vitro transcribed / translated 35S - Myc - CALCOCO1 and 35S - Myc - p62 with recombinant GST - GABARAPL2 ( WT and indicated mutants ) .

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C 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 ) .

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 .

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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 .

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.


HeLa 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 .

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HeLa 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 .

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 .

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

Immunoblot analysis of 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 (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 .

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

Immunoblot analysis of 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 .

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

, I 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 (

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

Immunoblot analysis of 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


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

Immunoblot analysis of 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.


B 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

O O O O O O O O O O O O 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 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 membrane


C , 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 .

C, 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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E HeLa cells transiently co-transfected with EGFP-CALCOCO1 and Myc-VAPA or -VAPB were immunostained with anti-Myc antibody. Scale bars, 5 μm.


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 .

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

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 analysis of HeLa cells transfected with the indicated siRNAs .

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 .

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

D, E 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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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 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 .

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

A, B 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 .

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

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 .

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

E 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 .

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

F, G 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 .

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 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 .

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

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 analysis of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 mLIR + ∆ 623 - 691 for 24 hours or not , and 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 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 .

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

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

D, 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 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 .

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

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 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 .

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

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 .

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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 - 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 .

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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 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 .

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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 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 .

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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 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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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 - 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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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 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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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 - 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

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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 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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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 - 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 .

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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 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 .

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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 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 .

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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 - 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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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 - 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 .

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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 cells 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.