CK-induced HK-1 cell death is dependent on AIF translocation from cytoplasm to nucleus. Cells were starved in 1% FBS supplemented RPMI 1640 medium for 1 day followed by treatment with CK (15 μM) for the indicated time intervals. (A) Cell lysates were collected and fractionated with nuclear extraction kit into cytosolic and nuclear fractions. Western blot analysis was used to determine the protein expression of AIF, lamin A/C (nuclear marker) and β-actin (cytosolic maker). (B) Immunostaining of cells was performed to show the translocation of AIF from cytoplasm to nucleus. Cells were stained with anti-AIF antibody and a PE secondary antibody (red) to localize AIF. DAPI staining (blue) was used to show the nuclei. Images were captured by confocal laser scanning microscope. Arrows indicate cells with translocated AIF. (C) Cytotoxic effect of CK on HK-1 was reduced by AIF siRNA. After transfection of siRNA, HK-1 cells were treated with CK (1 μM) for 24 h in serum-free RPMI 1640 medium, cell viability was determined by MTT assay and expressed as a percentage of solvent control in each experiment. Results were presented as mean ± SD from three independent experiments. **P < 0.01 compared to the DMSO control group, ##P < 0.01 versus non-targeting siRNA CK group.