Fig. 1

Metformin-mediated autophagy via the AMPK pathway. A HCT116 cells were treated with metformin (1, 5, and 25 mM) for 48 h. The levels of ATG5, p62, LC3-I, LC3-II, cleaved caspase-3, and PARP were examined by western blot analysis. β-Actin was used as a loading control in all western blot analyses. B HCT116 cells were treated with metformin (25 mM) for 48 h, followed by immunofluorescence analysis. The localization of LC3B (green) was visualized using a confocal microscope (original magnification ×1000, scale bar = 10 μm). (C) HCT116 cells were treated with metformin (1, 5, and 25 mM) for 48 h and then the levels of P-AMPK, P-ULK, P-mTOR, and P-p70 S6K were investigated by western blot analysis. D HCT116 cells were pretreated for 1 h in the presence or absence of Compound C (20 μM), followed by further treatment in the presence or absence of metformin (25 mM) for 48 h. The levels of P-AMPK, P-ULK, p62, 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 4PBA (5 mM) and then treated for 48 h in the presence or absence of metformin (25 mM). The levels of PERK, P-eIF2α, LC3-I, and LC3-II were investigated by western blot analysis. A–C Significance was determined by control and 25 mM metformin group Student’s t-test. **p < 0.01, ***p < 0.001