Fig. 1. Pantoprazole induced autophagosome biogenesis via TM9SF4-mTOR pathway.

a AGS cells treated with 120 μg/ml PPI for 48 h in pH 7.4 condition, were imaged by transmission electron microscopy. Representative micrographs showed increased abundance of lysosomes in cells after PPI treatment compared with control cells. Scale bar: 500 nm. b AGS and HeLa cells transfected with GFP-LC3B plasmid were treated with 120 μg/ml PPI for 48 h in pH 7.4 condition. Scale bar: 20 μm. c The number of autophagic vacuoles and GFP-LC3B dots in each cell were quantified. Data were presented as mean ± SD from three independent experiments (**p < 0.01, identified by Student’s t-test). d, e AGS and HeLa cells were treated for 48 h in pH 7.4 condition with indicated concentrations of PPI, or treated with 60 and 120 μg/ml PPI for the indicated times. Levels of LC3B-II and SQSTM1 protein were analyzed by western blot. β-actin served as an internal control. f mTOR pathway proteins levels in AGS cells after treated with PPI for 48 h both in pH 7.4 and 6.5 conditions, were detected by western blot analysis. g, h AGS cells were transfected with TM9SF4 siRNA for 48 h, and then incubated with 80 and 100 μg/ml PPI for another 48 h. The level of indicated proteins were analyzed by western blot (g). TM9SF4 knockdown efficiency was verified by Q-PCR (h). i Data showing the positive correlation between TM9SF4 and related autophagy genes mRNA expression in TCGA STAD corhort, were generated by GEPIA (http://gepia.cancer-pku.cn/detail.php?clicktag=correlation). Pearson’s coefficient tests were performed to assess statistical significance