Oncogenic Ras enhances the release of HtrA2/Omi from mitochondria into the cytosol. (A and B) NIH3T3 cells were infected with a control or Ha-RasV12–expressing retrovirus. (C–E) Ras-transformed NIH3T3 cells were transfected with a control or Flag-tagged Mfn2 expression vector and cultured for 36 h. (A and D) After subcellular fractionation of the cytosol (Cyto) and mitochondria (Mito), the distribution of HtrA2/Omi was evaluated by immunoblotting. COX IV and α-tubulin were used as mitochondrial and cytosolic markers, respectively. (B and C) Cells were transfected with a Mito-DsRed expression vector to visualize mitochondria. Images were obtained by three-dimensional structured illumination microscopy (top). Bars (main panels and insets), 10 µm. Colors indicate the lengths of mitochondria. Insets show the raw fluorescence images. Data represent the mean ± SEM (bottom); n = 5. (E) The cleavage of β-actin was evaluated by immunoblotting. Black arrowhead indicates full-length β-actin, and white arrowhead indicates a cleaved fragment.