Fig 2: RAB6 deficiency attenuated alveolar epithelial cell death in PM2.5-injured mice.a WT and RAB6−/− mice were intratracheally instilled with 50 μl of PM2.5 (100 mg/kg) or saline (instilled once a week, and continuously instilled four times). The apoptosis of alveolar epithelial cells in mice was determined by TUNEL staining (represented micrographs from six mice per group are shown, Scale bar = 100 μm). b Quantitative analysis of TUNEL positive cells. c The SFTPC+ AEC2 cells in lung tissue was detected by immunofluorescence staining (Blue, DAPI; red, SFTPC). (Represented micrographs from 6 mice per group are shown, Scale bar = 100 μm). d Quantitative analysis of the proportion of SFTPC+ AEC2 cells in lung tissue. e The ultrastructure of AEC1 cells and AEC2 cells in lung tissue was detected by transmission electron microscopy (TEM) (Nu nucleus; Mi mitochondrion; LB lamellar body; Ly lysosome; Va vacuoles) (Upper panel, scale bar = 10 μm; lower panel, scale bar = 0.5 μm). f The relative protein expression of RAB6, Bax, Bcl-2, and cleaved-caspase-3 in lung tissue was detected by western blot analysis. g Statistical analysis of relative expression levels of proteins. (n = 6; unpaired two-tailed t test. ***P < 0.001. Bar graphs represent the mean ± SEM for b, d, and g).

Fig 3: DKK1 inhibited proliferation and self-renewal of RAB6-/- AEC2 cells.a WT and RAB6−/− AEC2 cells were exposed to DKK1 protein (10 ng/ml) or PBS for 48 h and then cells were exposed to PM2.5 (100 μg/ml) for 48 h. The apoptosis of AEC2 cells was determined by FACS (Representative FACS images are shown, n = 3). b Statistical analysis of apoptosis rate. c The colony forming efficiency (CFE) of WT and RAB6−/− AEC2 cells was examined by plate colony formation assay (Representative images are shown, n = 3). d Statistical analysis of CFE (represented by colony numbers). e The relative protein expression of β-catenin, and c-myc in AEC2 cell lysates was detected by western blot analysis. f Statistical analysis of relative expression levels of proteins. g The relative protein expression of β-catenin in nuclear lysates of AEC2 cells was detected by western blot analysis. h Statistical analysis of relative expression levels of proteins. i–k The relative RNA expression of SOX2 (i), OCT4 (j), and NANOG (k) was measured by qRT-PCR. (n = 3; unpaired two-tailed t test. **P < 0.01; ***P < 0.001. Bar graphs represent the mean ± SEM for b, d, f, h–k).

Fig 4: A Rab6A-dependent immunosuppressive secretory process. (A) Correlation of Rab6A mRNA levels with percentages of total CD8+ T cells (A, Left) and exhausted CD8+ T cells (A, Right) quantified by flow cytometric analysis in a human LUAD cohort. (B) Flow cytometric analysis of Rab6A-deficient (shRab6A#3 and #4) or replete (shCTL) 344SQ flank tumors isolated from syngeneic, immunocompetent mice (n = 6 tumors per cohort). Percentages of T cells (CD8+ T cells, CD44low/CD62Lhigh naive CD8+ T cells, PD1+/TIM3+ exhausted CD8+ T cells and CD44high/CD62Llow effector/memory CD8+ T cells) and antigen-presenting cells (dendritic cells, total macrophages, M1 macrophages, and M2 macrophages) were quantified. (C and D) Flank tumor weight and lung metastasis numbers in syngeneic, immunocompetent mice (dots) injected with Rab6A-deficient (shRab6A#3 and #4) or replete (shCTL) 344SQ cells and treated with IgG CTL or anti-CD8 (C) or anti-CD80/CD86 antibodies (D). Fold-changes in tumor size and lung metastasis numbers were calculated for each cohort based on the mean values in anti-CD8-, anti-CD80/86, and IgG-treated groups. (E) Subcutaneous tumor growth curves in syngeneic, immunocompetent mice (dots). Cohorts are color-coded. Mean ± SEM. ***P < 0.0005. (F) CD8-, granzyme B-, CD11c-, and F4/80-positive cells in flank tumors generated by injection of 344SQ cells into syngeneic, immunocompetent mice. The percentage of positive cells in each cohort (plot). (G) Schematic illustration of a working model. ZEB1 relieves Rab6A, Rab8A, and their associated GEFs from miR-148a-dependent silencing, thereby accelerating Rab6A- and Rab8A-dependent exocytotic trafficking of vesicles carrying cargos that enhance FA turnover (e.g., MMP14) and establish an immunosuppressive TME (e.g., cytokines and ATX) to promote cancer metastasis.

Fig 5: Atg9-containing membranes adjacent to the TGN tubulates upon nutrient starvation in the presence of dynasore(A) HeLa/Atg9-GFP cells were pre-starved for 45 min to induce Golgi fragmentation. The cells were then incubated in starvation medium containing 80 μM Dynasore for 1 h, stained for TGN46 and analyzed by confocal microscopy. Magnified images in the dash-lined boxed area are shown in the right panels. (B) HeLa/Atg9-GFP cells were starved in the presence of 80 μM Dynasore for 1.5 h, stained for Rab6 and Bif-1, and analyzed by confocal microscopy. 3D Surface rendered (SR) images in the boxed area are shown in the right panels. (C) HeLa/S cells were starved in the presence of 80 μM Dynasore or control DMSO for 1.5 h and subjected to electron microscopy. Reticular structures resembling Golgi (orange) were not surrounded with vesicles (green) as observed in control DMSO-treated starved cells but detected nearby the tubular structures. In control starved cells (DMSO), endosome-like structures (yellow) were also detected around ER (pink)-associated immature autophagosome-like structures (blue). Scale bars represent 10 μm in A and B, and 2 μm in C and the insets in A and B.