Figure 7.
Figure 7. Oncogenic Ras increases mitochondrial translocation of p38 MAPK and phosphorylation of HtrA2/Omi in a p53-dependent manner. (A–E) NIH3T3 cells and p53−/− MEFs were infected with a control or Ha-RasV12–expressing retrovirus. (F) NIH3T3 cells were infected with a Ha-RasV12–expressing retrovirus together with a control or p53R175H-expressing retrovirus. (G and H) Ras-transformed NIH3T3 cells were treated with 10 µM BIRB796 for 16 h. (A) p53−/− MEFs were transfected with a Mito-DsRed expression vector to visualize mitochondria. Images were obtained by three-dimensional structured illumination microscopy (top). Bars (main panel and inset), 10 µm. Colors indicate the lengths of mitochondria. Insets show the raw fluorescence images. Data represent the mean ± SEM (bottom); n = 5. (B) Confocal images of cells stained for HtrA2/Omi. Bars (top), 10 µm. The square region in the top panel is shown magnified in the bottom panel. Bars (bottom), 1 µm. (C) Relative fluorescence intensities in the cytosol were calculated from the images in B. Data represent the mean ± SD of more than 15 cells. *, P < 0.01. (D) The distribution of HtrA2/Omi and p38 MAPK, and the levels of phosphorylated HtrA2/Omi (p-HtrA2) and phosphorylated p38 MAPK (p-p38), were evaluated by immunoblotting after subcellular fractionation of the cytosol (Cyto) and mitochondria (Mito). COX IV and α-tubulin were used as mitochondrial and cytosolic markers, respectively. (E and F) The levels of phosphorylated HtrA2/Omi and phosphorylated p38 MAPK were evaluated by immunoblotting. Black arrowheads indicate phosphorylated HtrA2/Omi. The level of phosphorylated HtrA2/Omi in the mitochondrial fraction was also evaluated. (G) The level of phosphorylated HtrA2/Omi in the mitochondrial fraction was evaluated by immunoblotting. (H) The cleavage of β-actin was evaluated by immunoblotting. Black arrowhead indicates full-length β-actin, and white arrowhead indicates a cleaved fragment. The relative amount of the cleaved fragment of β-actin to the total amount of β-actin was quantified.

Oncogenic Ras increases mitochondrial translocation of p38 MAPK and phosphorylation of HtrA2/Omi in a p53-dependent manner. (A–E) NIH3T3 cells and p53−/− MEFs were infected with a control or Ha-RasV12–expressing retrovirus. (F) NIH3T3 cells were infected with a Ha-RasV12–expressing retrovirus together with a control or p53R175H-expressing retrovirus. (G and H) Ras-transformed NIH3T3 cells were treated with 10 µM BIRB796 for 16 h. (A) p53−/− MEFs were transfected with a Mito-DsRed expression vector to visualize mitochondria. Images were obtained by three-dimensional structured illumination microscopy (top). Bars (main panel and inset), 10 µm. Colors indicate the lengths of mitochondria. Insets show the raw fluorescence images. Data represent the mean ± SEM (bottom); n = 5. (B) Confocal images of cells stained for HtrA2/Omi. Bars (top), 10 µm. The square region in the top panel is shown magnified in the bottom panel. Bars (bottom), 1 µm. (C) Relative fluorescence intensities in the cytosol were calculated from the images in B. Data represent the mean ± SD of more than 15 cells. *, P < 0.01. (D) The distribution of HtrA2/Omi and p38 MAPK, and the levels of phosphorylated HtrA2/Omi (p-HtrA2) and phosphorylated p38 MAPK (p-p38), were evaluated by immunoblotting after subcellular fractionation of the cytosol (Cyto) and mitochondria (Mito). COX IV and α-tubulin were used as mitochondrial and cytosolic markers, respectively. (E and F) The levels of phosphorylated HtrA2/Omi and phosphorylated p38 MAPK were evaluated by immunoblotting. Black arrowheads indicate phosphorylated HtrA2/Omi. The level of phosphorylated HtrA2/Omi in the mitochondrial fraction was also evaluated. (G) The level of phosphorylated HtrA2/Omi in the mitochondrial fraction was evaluated by immunoblotting. (H) The cleavage of β-actin was evaluated by immunoblotting. Black arrowhead indicates full-length β-actin, and white arrowhead indicates a cleaved fragment. The relative amount of the cleaved fragment of β-actin to the total amount of β-actin was quantified.

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