Figure 5

Pharmacologic inhibition of autophagy reduces metformin-mediated apoptotic cell death. (A) Ishikawa cells were seeded in 96-well plates and incubated with the indicated dose of metformin (with or without 3MA or CQ) for 48 h; cell viability was determined by WST-8 assays. Data are presented as the mean ± SD of three independent replicate measurements. ^*p < 0.05 and ^**p < 0.01 vs. 3MA or CQ untreated cells. (B) Flow cytometry of apoptosis in Ishikawa cells treated with metformin (with or without 3MA) for 48 h. ^*p < 0.05 and ^**p < 0.01 vs. 3MA untreated cells. (C) Ishikawa cells were incubated with the indicated dose of metformin alone or combined with 3MA for 48 h. Cells were then lysed to measure caspase-3/7 activity. *p < 0.05 and **p < 0.01 vs. 3MA untreated cells. (D) Ishikawa cells were treated with the indicated dose of metformin (±3MA) for 48 h, and cell lysates were subjected to western blot analysis using antibodies against LC3B, p62, and cleaved PARP. Actin was used as a loading control.