Fig. 5. P35 inhibits mitochondrial cytochrome c release, mitochondrial depolarization and caspase activation. RAW 264.7 cells or vector- or P35-transfected clones (1x10⁶ cells/well) in 6-well tissue culture plates were treated with cisplatin (2 µg/ml) and IFN- (25 U/ml), or LPS (1 µg/ml) and IFN- (25 U/ml) in the presence or absence of L-NMMA (1 mM). Cells were also treated directly with NO donor, SNP (1 mM). Cytochrome c in the cytosol and mitochondria was detected by immunoblot analysis (A). Mitochondrial depolarization was determined as described in Materials and Methods, and results were expressed as the percentage of cells with depolarized mitochondria (B). Extracts were prepared as described in Materials and Methods. Caspase-9 (C) and caspase 3 (D) activities were measured in protein extracts as described in Materials and Methods. Data shown are mean±s.e.m. and are representative of three independent experiments done in triplicate. ^*P<0.05 versus values of control cultures. ^**P<0.05 versus values of cisplatin and IFN-/LPS and IFN--treated cultures. ^#P<0.05 versus values for cisplatin and IFN-/LPS and IFN- or SNP-treated RAW 264.7/pCMV-FLAG cells.