ROS production is required for AIF activation in HepG2 cells. (A, B) Cells were treated with matrine at different concentrations (0, 0.25, 0.5, 1, 1.5, 2 mg/ml) for 24 hrs or at 1.5 mg/ml for 24 hrs in the absence or presence of Tiron (5 μM), the ROS scavenger. ROS levels were then detected by flow cytometry. (C) Tiron significantly attenuated matrine-induced cell death as determined by Annexin V/PI staining and flow cytometry. The basal level of cell death was normalized to 1.*p < 0.05, **p < 0.01 vs. Matrine. Immunostaining (D) and western blot analyses (E) demonstrated that matrine-induced AIF translocation to the nucleus was inhibited by Tiron.