Fig 2: miR-21 regulates the autophagy of colorectal cancer cells through PTEN/AKT/TFEB.a Autophagic structures (AV, AL, and IM) were observed in parental RKO and RKO-miR-21-KO cells by transmission electron microscope. Number of autophagic structures was counted. b RKO and RKO-miR-21-KO were treated with glucose starvation or bafilomycin A1, the LC3 (LC3I and LC3II) expression was measured by Western blot. c The protein expression level of PTEN, phosphorylated AKT (Ser473), VMP1, LC3, and nuclear TFEB were measured by Western blot in RKO and RKO-miR-21-KO cells. d RKO and RKO-miR-21-KO were treated with VO-Ohpic at concentrations of 1–4µM for 24 h, and immunoblot analysis of phosphorylated AKT (Ser473), VMP1, LC3 expression and the protein amount of nuclear TFEB. e RKO and RKO-miR-21-KO were treated with Afuresertib at concentrations of 1–4 µM for 24 h, and immunoblot analysis of VMP1, LC3 expression, and the protein amount of nuclear TFEB. f The protein expression level of VMP1, LC3, total TFEB, and the protein amount of nuclear TFEB were measured by Western blot after knock-down of TFEB expression by siRNA in RKO and RKO-miR-21-KO cells. (Scale bars, 0.5 µm) Data are presented as mean ± SD. **P < 0.01.

Fig 3: VMP1 is associated to ER sliding events.(A) HeLa and Cos-7 cells transfected with a plasmid encoding VMP1 fused to GFP (VMP1-GFP) and visualized by confocal microscopy show a similar pattern. (B) Analysis of confocal microscopy of Cos-7 cells expressing VMP1-GFP and the ER marker Sec61. The punctated pattern is evident in the peripheral tubules and the perinuclear area. (C) In vivo time-lapse confocal microscopy of cells expressing VMP1-GFP and Sec61-mCherry. Different examples of sliding events are shown in frames. The corresponding movies are available as supplementary material (S1–S3 Movies). (D) ΔNt-VMP1-GFP were transfected in HeLa cells and colocalized with the ER marker Sec61 by confocal microscopy. The protein distributes throughout the ER loosing the punctated pattern in HeLa cells.

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: 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)