IH-induced apoptosis is mediated by AIF translocation instead of caspase-3 activation. Aa, b: Pictures and quantitative assessment of staining of the caspase-3-cleaved substrate FITC-DEVD-FMK. There was no significant difference between the RA and IH groups. B: Western blotting analysis of whole cell proteins showed no activated caspase-3 or cleaved PARP. Ca: Cells were costained with AIF and VDAC. N: nucleus; M: mitochondria. Cb: Quantitative assessment of AIF staining based on the ratio of N/M fluorescence. D: Western blotting analysis of subcellular fractions. The mitochondria did not release cytochrome c, and activated caspase-3 was not noted. AIF was released from the mitochondria in the IH4day group. β-actin was used as an internal control. VDAC was used as a loading control of mitochondrial fraction. M: marker. *p < 0.05 for comparing each group with IH4day group by Dunnett's test in ANOVA.