Fig. 4. Mitochondrial permeability changes during apoptotic cell death in ARPE-19 cells. (A) ARPE-19 cells, treated with 50 µM menadione for 4 hours were incubated with CMXRos, fixed, immunolabeled with anti-AIF antibodies and put on a coverslip with DAPI-containing mounting media. In control cells (top), immunostaining of AIF colocalized with CMXRos and was excluded from the DAPI-labeled nuclei. In menadione-treated cells (bottom), AIF is colocalized with DAPI-labeled nuclei, and CMXRos staining is diminished. Bar, 10 µm. (B) Redistribution of AIF and cytochrome c during ARPE-19 cell death. Whole cells or subcellular extracts of control or ARPE-19 cells treated with 50 µM menadione for 4 hours were analyzed for AIF and cytochrome c translocation using western blot analysis. Actin was used to normalize whole cell and cytosolic loading. Cytochrome c oxidase II and Oct-1 were used for normalization of mitochondrial and nuclear fraction loading, respectively. (Data represent two independent experiments). (C) The values are the percentage of ARPE-19 cells treated as in A displaying nuclear AIF and loss of CMXRos staining obtained by averaging three fields per slide in which approximately 150 cells per slide were counted. The results shown are mean±s.d., which are representative of two independent experiments. (*, P<0.001, ANOVA/t-test, unpaired).