Figure 5
Inactivation of Stat3/Bcl-2 signaling contributes to metformin-induced effects. (a) Metformin treatment repressed expression of p-Stat3 and cyclin D1, as assayed by western blot. (b) Western blotting was used to assess p-Stat3, Stat3 targets (cyclin D1 and Bcl-2), apoptosis-related proteins, and autophagy-related proteins in Stat3 siRNA – or control siRNA-transfected EC109 cells treated with metformin. (c) Representative images ( × 1000) of GFP-LC3 expression patterns in Stat3 siRNA and control siRNA cells following metformin treatment. EC109 cells transfected with GFP-LC3 plasmid were used. (d) Ectopic expression of Bcl-2 attenuates metformin-mediated autophagy and apoptosis, as indicated by related protein expression assayed by western blot. (e) Representative images of IHC staining of p-Stat3 and Bcl-2 in adjacent nontumor ( × 200) and ESCC tumor tissues ( × 400) from patients. The p-Stat3 protein level positively correlated with the Bcl-2 protein level in ESCC tumors from 36 patient samples (Pearson’s correlation coefficient test, r=0.695 and P<0.001). (f) Effects of metformin treatment (10 mM for 48 h) on expression of AMPK/mTOR, p-Stat3 and LC3, as assayed by western blot, following transfection with AMPK siRNA or control siRNA. Representative data from one of three independent experiments are shown in (a, b, c, d and f). β-Actin was applied as the loading control for (a, b, d and f)
