Matrine induced caspase-dependent and -independent cell death in HepG2 cells. (A) The chemical structure of matrine. (B) Cells were treated with different concentrations of matrine (0, 0.25, 0.5, 1, 1.5, 2 mg/ml) for 24 hrs, or 1.5 mg/ml matrine for different time periods (0, 12, 24, 36, 48 hrs), and then the cell death was determined by Annexin V/PI staining assay. (C) Cells were collected after treated with different concentrations of matrine for 24 hrs and analyzed for ΔΨm by Rh123 retention. (D) Expression of cytosolic and mitochondrial cytochrome c in HepG2 cells with matrine treatment. Cells were treated with 1.5 mg/ml of matrine for 24 hrs. The cytoplasmic and mitochondrial proteins were segregated and analyzed by western blot assay. (E) HepG2 cells were treated with different concentrations of matrine for 24 hrs. The total protein was extracted and the expression of apoptosis-related proteins was analyzed by western blot assay. (F) Effect of pancaspase inhibitor, z-VAD-fmk, on matrine-induced cell death. HepG2 cells were pretreated with z-VAD-fmk (20 μM, 2 hrs) before matrine treatment. Cell death was analyzed by Annexin V/PI staining assay. Data are presented as means ± S.D. of three separate experiments. The basal level of cell death was normalized to 1. *p < 0.05, **p < 0.01 vs. control.