EMBO/SourceData huggingface dataset:
Figure 3(A-C) (A) ABCA1, (B) ABCG1 and (C) LXRβ expression in islets of 8 week old mice assessed by Western blotting (mean±SEM, n=3-6 mice per genotype, relative to β-actin controls, *p<0.05, **p=<0.01, Students t-test).(D) Western blot of cell lysates of MIN6 cells after silencing of Abca12, probed with antibodies against ABCA1, ABCG1, SR-B1 and LXRβ.(E) Total cholesterol (COH) or cholesteryl esters (CE) levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β-cells (mean±SEM n=9, 5 and 5 mice per genotype respectively).(F) Ceramides levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β-cells (mean±SEM n=10, 4 and 5 mice per genotype respectively).(G) Lipid levels in MIN6 cells in which ABCA12 had been depleted by siRNA treatment (mean±SEM, n=5 (biological replicates assayed in triplicate), FC = free cholesterol, CE = cholesterol ester, Cer = ceramide, SM = sphingomyelin, TG = triacylglycerol, PC = phosphatidylcholine, *p<0.05, **p=<0.01, Students t-test).(H) Cholesterol efflux to lipid-free apoA-I (30 μg/ml, 2 h) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 (4 μM)(mean±SEM, n=4, (biological replicates assayed in triplicate), *p=<0.05, Students t-test).(I) Cholesterol efflux to HDL (30 μg/ml, 2 h) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 (4 μM); (mean±SEM, n=4 (biological replicates assayed in triplicate)).(J) The effect of overexpression of ABCA1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test), inset shows a Western blot of cell lysates probed with anti-ABCA1 antibody in which the order of bands corresponds to the order of bars).(K) The effect of overexpression of ABCG1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test, inset shows a Western blot of cell lysate probed with anti-ABCG1 anitbodies in which the order of bands corresponds to the order of bars).(L) The effect of overexpression of ABCA1 and ABCG1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test).(M) The effect of activation of cells with TO901317 (4 μM) on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test).(N) The effect of overexpression of LXRβ on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates), *p=<0.05, Students t-test, inset shows a Western blot of cell lysates probed with anti-LXRβ antibody in which the order of bands corresponds to the order of bars).
Figure 4(B) Scatter plot and heat map of differential gene expression between cre control and Abca12tm1d mice at 8 weeks of age. Legend indicates Log2 fold change values.(C) Circulating glucose levels after administration of intraperitoneal L-arginine in mice at 8 weeks of age, mean±SEM, n =3 mice per genotype, *p=<0.05 of cre relative to tm1d, Students t-test).(D) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 15mM glucose (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the lower panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01, ***p=<0.001 of cre relative to tm1d, Students t-test).(E) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 20mM KCl (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the right panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01 of cre relative to tm1d, Students t-test).(F) Assessment of calcium flux measured by Fluo4 in response to either Glucose or Potassium in isolated islets from 11 week old animals. Fluorescence responses were corrected for basal fill (F/F0). (mean±SEM, each data point represents the average of 10 cells/coverslip for 13-16 coverslips for each of 4 wild type and 5 Abca12tm1d mice)(G) Expression of GLUT2 in isolated mouse islets at 8 weeks of age. The upper panel depicts a representative Western blot and the lower graph the quantitation of protein levels from 3 mice of each genotype, normalised to β-actin (mean±SEM).(H) Insulin content of islets of Abca12tm1d mice (mean±SEM, n=3 mice of each genotype, normalised to total protein content).
Figure 5(A-C) Transmission electron microscopy of β-cells from Abca12tm1d and control mice at 8 weeks of age. Panels A'-C' depict higher resolution images from the boxed regions in A-C (scale bar = 1μm).(D) Quantitation of the area of 3000 granules from each genotype (mean±SEM, n=4, 3 and 3 animals respectively, *p=<0.05 of cre relative to tm1d, Students t-test). (E) Relative frequency distribution of granule areas from (D). (F) Assessment of total cholesterol (COH) by mass spectroscopy in mice fed normal chow or a high cholesterol diet (HCD)(mean±SEM, n=5 and 4 animals respectively, **p=<0.01, Students t-test).(G, H) Quantification of change in mean granule area in Abca12tm1d on chow diet and Abca12tm1d on high cholesterol diet islets assessed by transmission electron microscopy (n=3000 granules/genotype, mean±SEM, n=4, 3, 3 and 4 animals respectively, *p=<0.05, Students t-test) as an average and as a relative frequency distribution.(I) Correction in abnormal granule shape following high cholesterol feeding (bar = 1μm), with I' showing magnification of the boxed region indicated.(J) Glucose tolerance test results following intraperitoneal glucose challenge in Abca12tm1d and cre control mice fed normal chow or HCD (mean±SEM, n=3 mice per genotype, *p=<0.05, for tm1d versus tm1d+HCD, Students t-test).(K) Serum insulin in Abca12tm1d +/- HCD mice after i.p glucose challenge (mean±SEM, n=3 mice/genotype, **p=<0.01, Students t-test).
Figure 6(A) Detection of lipid rafts in 8 week old islets with recombinant cholera toxin subunit B (CT-B) conjugated to Alexa Fluor 488 (green). Islets are co-stained with DAPI (blue) (scale bar = 50μm.).(B) FACS profiling of dispersed islets from 12 week old mice stained with insulin and CT-B.(C) Confocal images of rafts (using CTB-Alexa Fluor 647) in MIN6 cells transfected with Fluorescein-488-labelled siRNAScr or Dy547-labelled siRNAABCA12 (scale bar=10μm). (D) Quantitation by confocal microscopy of the effect of ABCA12-deficincy on CTB binding to MIN6 cells (mean±SEM, *p=<0.05, Students t-test, n=4 biological replicates, 50-100 cells for each). (E) Quantitation by flow cytometry of the effect of ABCA12-deficiency on CTB binding to MIN6 cells (mean±SEM, n=3 biological replicates, *p=<0.05, Students t-test). (F) The effect 10 mM MβCD on GSIS in MIN6 cells depleted or not for ABCA12 (incubation for 5 or 10 minutes as indicated, mean±SEM, n=4 (biological replicates), *p=<0.05, Students t-test).(G) Western blot of the abundance of CDC42 in ABCA12 deficient MIN6 cells.(H) The effect of ABCA12 deficiency on activation of CDC42 by bradykinin in MIN6 cells (BDK, 100 ng/ml, 4 min; *p=<0.05, Students t-test, significance versus ABCA12-deficient BDK activated cells is indicated, n=4 biological replicates, mean±SEM).(I,J) (I) Confocal images of F-actin in ABCA12-deficient MIN6 cells (scale bar = 20μm) and (J) quantification of abundance showing the effect of ABCA12-deficiency (mean±SEM, *p=<0.05, Students t-test, n=4 biological replicates, 50-100 cells for each).(K) F-actin levels assessed by FACS sorting of labelled MIN6 cells transfected with siRNAABCA12 (mean±SEM, n=3 biological replicates, *p=<0.05, Students t-test).(L,M) (L) Representative image of F-actin using LifeAct staining of purified islets isolated from Abca12tm1d versus control mice (scale bar = 10μm) and (M) quantitation of fluorescent signals from these samples (n=3 biological replicates, mean±SEM, ***p=<0.001, ****p=<0.0001 Students t-test).(N) Colocalization between ABCA12 (red) and CDC42 (green) in MIN6 cells (scale bar = 10μm).O) Effects of overexpression of a constitutively active form of CDC42 on GSIS from MIN6 cells with ABCA12-deficiency (mean±SEM, n=4 biological replicates, *p=<0.05, Students t-test).(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).(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)
Figure 7(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).(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).(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).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).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).
Figure 1B) 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. 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.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.
Figure 2A) 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.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).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.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.
Figure 3A) 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.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.
Figure 4A) 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.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.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 DMSOF) 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.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.
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).
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.