Figure 5.

Antcin C protects HepG2 cells from AAPH-induced apoptosis. (a) The DNA fragmentation in AAPH-induced HepG2 cells was determined by TUNEL assay as described in Materials and Methods. The average number of TUNEL-positive cells in microscopic fields (magnification ×200) from three separate samples. (b) Percentages of apoptotic cells were determined by the number of TUNEL positive cells compared to the control. Values represent the mean ± SD of three independent experiments. ***P < 0.001 was considered significant for AAPH versus sample. ^† P < 0.001 was considered significant for control versus AAPH. ((c), (e), (f)) HepG2 cells were pretreated with antcin C (20 μM) for 2 h, and then exposed to AAPH for 30 min. The effects of antcin C on AAPH-induced cytosolic cytochrome c, caspase 9, caspase 3, PARP, Bcl-2, and Bax (mitochondrial pathway) protein levels were examined by immunoblotting. (g) Caspase 4, caspase 12, and HSP70 (ER stress pathway) was examined using western blot analysis. (d) Cells were pretreated with antcin C with or without caspase-3 inhibitor Z-VAD-FMK (30 μM) for 2 h, and then cell death was induced by AAPH for 24 h. Cell viability was determined by MTT assay. Values represent the mean ± SD of three independent experiments. **P < 0.01 was considered significant for AAPH versus samples. ^† P < 0.01 was considered significant for control versus AAPH.