Fig. 2

Enhancement of autophagic flux by blockade of O-GlcNAcylation. A HCT116 cells were incubated with metformin (1, 5, 10, 20, and 25 mM) for 48 h, and cell viability was determined by MTT assay. B HCT116 cells were treated with metformin (1, 5, and 25 mM) for 48 h, the levels of O-GlcNAc and GFAT were analyzed by western blot. β-Actin was used as a loading control in all western blot analyses. C HCT116 cells were cultured in the presence or absence of Thiamet G (80 μM) or OSMI-1 (20 μM) and then treated with or without metformin (25 mM) for 48 h. The levels of LC3-I and LC3-II were determined by western blot analysis. D HCT116 cells were treated with various concentrations of OSMI-1 (5, 10, and 20 μM) for 48 h, and the levels of IRE1α, LC3-I, and LC3-II were investigated by western blot analysis. E HCT116 cells were cultured for 1 h in the presence or absence of 4µ8c (20 µM) and then treated for 48 h with or without OSMI-1 (20 µM). The levels of IRE1α, LC3-I, and LC3-II were investigated by western blot analysis (top). Levels of sXBP1 were determined by RT-PCR using primers to amplify both unspliced and spliced mRNA species (bottom). 18 s rRNA was used as the loading control for RT-PCR. A, D Significance was determined by control and 20 μM OSMI-1 group Student’s t-test. ***p < 0.001