Bid mediated the cell death induced by matrine, as an upstream regulator of AIF. HepG2 cells were transfected with Bid siRNA (40 or 60 nM) or non-targeted siRNA for 24 hrs (A, C, E) or pretreated Bid inhibitor BI-6C9 (10 μM) for 1 hr (B, D, F), then treated with matrine at 1.5 mg/ml for 24 hrs. (A, B) Immunostaining was performed to localize AIF with an anti-AIF antibody and a FITC-conjugated secondary antibody (green). The nuclei were detected using DAPI staining (blue). (C, D) Cytoplasmic, nuclear, and mitochondrial fractions were prepared for western blot analysis to localize AIF with an anti-AIF antibody using anti-AIF, anti-β-tubulin (cytosolic marker), anti-Hsp60 (mitochondrial marker) and anti-Lamin B (nuclear marker) antibodies. (E, F) Cells were stained with Annexin V/PI to assess cell viability by flow cytometry. The basal level of cell death was normalized to 1.*p < 0.05, **p < 0.01 vs. non-target siRNA + Matrine (E), Matrine (F).