Fig. 5: DDX17/ MAGEA6/AMPKα1 signaling regulates autophagy in LUAD cells.

A, B Level of LC3 protein by western blotting in DDX17-knockdown cells (A), or DDX17-overexpression cells (B), and the corresponding control cells. C H1299-shDDX17#2 cells or control cells were transiently transfected with mCherry-EGFP-LC3 reporter plasmids, and analyzed by IF assays. Yellow signals represented the non-acidic autophagosomes and red signals represented the acidic autophagolysosomes in merged images (Top panels). Scale bars = 10 μm. Bottom panels: quantitative analysis of red dots / yellow dots of the merged images in cells (mean ± s.e.m. of counted cells, n ≥ 12 cells per cohort, determined by two-tailed Student’s t-tests). D Representative photos of IF staining of LC3 (red) puncta in H1299-shDDX17#2 cells or control cells (left panel). Scale bars =10 μm. The right panel is the statistical analysis of LC3 puncta (mean ± s.d. of counted cells, n  ≥ 12 cells per cohort, determined by two-way ANOVA). E MTT assays were performed in DDX17 silencing or control H1299 cells (left) treated with or without 3-MA (1 mM), and A549 cells (right) treated with or without 3-MA (0.25 mM), respectively. Data are presented as mean ± s.e.m. from three independent experiments. *p < 0.05; ns, p > 0.05, determined by two-way ANOVA. F Western blotting showing the level of MAGEA6 and AMPKα1 in DDX17-knockdown cells, or DDX17-overexpression cells. G AMPKα1 level in H1299-shDDX17 #2 cells or control cells with or without MAGEA6 stable overexpression. H LC3 levels in H1299-shDDX17 #2 cells or control cells with or without MAGEA6 stable overexpression. I Representative photos of IF staining of LC3 (red) puncta in H1299-shDDX17 #2 cells or control cells with or without MAGEA6 stable overexpression. J Statistical analysis of LC3 puncta in I (mean ± s.e.m. of counted cells, n ≥ 12 cells per cohort, determined by two-way ANOVA).