AIF translocation is required for matrine-induced ciPCD. (A-B) HepG2 cells were transfected with AIF siRNA (40 or 60 nM) or non-targeted siRNA for 24 hrs, and then treated with matrine at 1.5 mg/ml for 24 hrs. (A) AIF expression levels were detected by western blot analysis using an anti-AIF antibody. Actin was used as a protein loading control. (B) 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. (C) 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). (D) Cytoplasmic, nuclear, and mitochondrial fractions were prepared for western blot analysis using anti-AIF, anti-β-tubulin (cytosolic marker), anti-Hsp60 (mitochondrial marker) and anti-Lamin B (nuclear marker) antibodies.