Fig. 8. Influence of EGF on the cytochrome c release from mitochondria. (A) The SKBr3/EGFR cells were incubated in the presence of EGF (20 ng/ml) or vehicle for indicated times. The cells were then homogenized and subjected to subcellular fractionation and fractions containing mitochondria or cytosol were analyzed by western blot (W.B.) for the presence of cytochrome c. The cytosol fractions are about 50 times more dilute than the mitochondrial fractions, as indicated in the Materials and Methods. (B) A431/ErbB2-mito cells were treated with EGF for 48 hours, fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and stained with cytochrome c antibody. The two thinner arrows indicate cells that show colocalization of cytochrome c (green) with the mitochondrial network (red). A thick arrow indicates a cell with cytochrome c released in the cytosol. Notably, this cell has a compressed mitochondrial network still entrapping most of the cytochrome c. Bar, 20 µm.