A, dual immunofluorescence of cytochrome c and active Bax in cisplatin-treated cells. Kidney tubular cells were incubated with 20 μm cisplatin and fixed for dual immunofluorescence of cytochrome c and active Bax. Arrows: cells with active Bax and cytochrome c release. Asterisks: cells without active Bax and cytochrome c release. B, immunoblot analysis of Bax translocation to mitochondria. C, immunoblot analysis of cytochrome c release. Kidney tubular cells were incubated with 20 μm cisplatin in the absence or presence of 20 μm Nutlin-3 for 24 h. The cells were fractionated into cytosolic (Cytosol) and membrane-bound organellar fractions containing mitochondria (Organelle) for immunoblot analysis of Bax and cytochrome c. β-Actin and cytochrome oxidase IV (CoxIV) were used as internal protein loading controls for cytosolic and membrane fractions, respectively. The results show Bax activation and translocation to mitochondria during cisplatin treatment, accompanied by cytochrome c release. Both Bax activation and cytochrome c release were suppressed by Nutlin-3.