Figure 4
Inhibition of autophagy sensitizes cells to metformin-induced apoptotic cell death. (a) Inhibition of autophagy with 3-MA or CQ decreases viable metformin-treated cells. Cell viability was measured by MTT assay after cells were incubated with the indicated concentration of metformin with or without 3-MA (±3-MA, 5 mM; ±CQ, 25 μM) for 48 h (left and middle). Cell viability was measured by MTT assay after cells were incubated with the indicated concentration of 3-MA with or without metformin (±metformin, 10 mM) for 48 h (right). Data were expressed as the means±SD from three separate experiments as the percentage of viable cells in the control group. *P<0.05 and **P<0.01 by Student’s t-test. (b) Representative images of TUNEL staining of EC109 cells after treatment with the indicated concentration of metformin with or without 3-MA (±3-MA) for 48 h (magnification, × 400). Quantification of the number of TUNEL-positive cells (graph on the right), plotted as a percentage TUNEL-positive cells. **P<0.01 by Student’s t-test. (c) Cells were stained with JC-1 (10 μg/ml) following metformin treatment with or without metformin CQ (±CQ) for 48 h. Mitochondrial membrane potential (MMP) was analyzed using flow cytometry. (d) Apoptosis- and autophagy-related proteins were examined by western blot following metformin treatment with or without 3-MA (±3-MA) for 48 h. (e) Effects of genetic inhibition of autophagy by knockdown of Beclin-1 or Atg5 on metformin-mediated apoptosis. LC3 and cleaved PARP were examined by western blot. Representative data from one of three independent experiments are shown in (b–e). Error bars indicate±S.E. *P<0.05 and **P<0.01 by Student’s t-test. β-Actin was the loading control in (d) and (e)
