Fig 2: Inhibition of VMP1 palmitoylation abolished the interaction between VMP1 and ALIX. A SCs were transfected with WT-VMP1-flag, MT-VMP1-flag or flag-tagged control plasmid (NC), and then conducted immunoprecipitation with anti-Flag. Total lysates and flag immunoprecipitates were then blotted. B Venn diagram of mass spectrometry results showed proteins that interacted with Flag tagged WT-VMP1, MT-VMP1, NC vectors. C Double-immunofluorescence of endogenous VMP1 and ALIX expression in SCs. The co-localization area is magnified in white box. D SCs were transfected with His-ALIX and WT-VMP1 or MT-VMP1, and tested for the interaction of exogenous VMP1 and ALIX by immunoprecipitation with anti-His antibody. β-actin served as a loading control. E The western blot of exosomal marker protein CD63, ALIX, TSG101 in sEVs obtained from SCs transfected with WT-VMP1 or MT-VMP1 and siALIX or siNC vector. F Quantification of relative exosomal protein CD63, ALIX, TSG101 in E of three independent experiments. Data were presented as mean ± SEM, *P < 0.05. ns: not significant. G Double immunofluorescence of CHMP4 and ALIX expression in SCs transfected with WT-VMP1 or MT-VMP1 vector. Right panel: Statistical analysis of the co-localization rate between ALIX and CHMP4 in the two groups. The experiment was repeated three times

Fig 3: VMP1 is an ER protein in close proximity to vesicles and mitochondria.Cells expressing VMP1-GFP were subjected to inmuno-gold labeling with an antibody against GFP and visualized by TEM. Labeling is found in nuclear membrane (NM) (A) and ER tubules (B) as well as in close proximity to vesicles (C) and mitochondria (M)

Fig 4: Gemcitabine induces E2F1 activation of VMP1-mediated autophagy only in pancreatic tumor cells that carry oncogenic KRAS. qPCR and western blot analysis showed no significant differences of VMP1 mRNA (A) and protein expression (B) in BxPC-3 treated with 20 μM gemcitabine for 12 and 24 h (n = 3). (B) Western blot analysis shows no difference of VMP1 expression and LC3 lipidation in BxPC-3 cells submitted to gemcitabine treatment. The use of chloroquine alone or in combination with gemcitabine treatment induced LC3-II accumulation compared to control cells or cells only treated with gemcitabine in the same proportion, respectively, indicating that gemcitabine does not interrupt autophagy flux in these cells. (C) Relative densitometry (VMP1/Actin and LC3-II/Actin) of western blots shown in B (n = 3). (D) Western blot analysis shows that E2F1 expression was increased in PANC-1 cells submitted to gemcitabine treatment for 12 and 24 h, and there were no significant differences in the expression of E2F1 in BxPC-3 cells. Relative densitometry (E2F1/Actin) of western blots is shown (n = 3). PANC-1 cells were transiently transfected with E2F1 expression vector or empty vector as control. (E) qPCR assay demonstrated an increase of VMP1 mRNA expression in PANC-1 cells expressing E2F1 compared to control cells (n = 3). (F) E2F1 expression induced autophagy as was determined by counting cells with punctate RFP-LC3 staining. Representative images of RFP-LC3 distribution using confocal microscopy are shown (bar scale = 10 μm). The graph presents the percentage of RFP-LC3 cells with punctate staining (n = 3). *p < 0.05, **p < 0.01.

Fig 5: Palmitoylation affects VMP1 subcellular localization. A Analysis of surface expression by biotinylation. HEK293T cells expressing WT-VMP1, MT-VMP1(C8, 192S), or (C263, 278S) treated with ethanol (NC) or 2-BP, and Sertoli cells expressing WT-VMP1 or MT-VMP1 were used for surface biotinylation assays. Total and surface proteins were analyzed by western blot using anti-flag antibody. B Sertoli cells overexpressing WT-VMP1 or MT-VMP1 were double-immunostained with anti-Flag antibody and the antibody of Na+/K+-ATPase, the marker of plasma membrane (PM). This experiment was repeated three times independently. C Statistical analysis of the colocalization rate between WT-VMP1/ MT-VMP1-flag and Na+/K+-ATPase in SCs. Values are means ± SEM from n = 3 independent experiments. *P < 0.05, the P value was determined by two-sided Student’s t-test