Figure 4.
Figure 4. Knockdown of HtrA2/Omi attenuates the cleavage of β-actin and restores p130Cas phosphorylation in Ras-transformed cells. (A and B) NIH3T3 cells, iMEFs, and p53−/− MEFs were infected with a control or Ha-RasV12–expressing retrovirus. (C and D) NIH3T3 cells were infected with a Ha-RasV12–expressing retrovirus together with a control or p53R175H-expressing retrovirus. (E) NIH3T3 cells were infected with a control or Ha-RasV12–expressing retrovirus together with a control or HtrA2/Omi shRNA–expressing retrovirus. (F, and I–M) NIH3T3 cells were infected Ha-RasV12–expressing retrovirus and a control or HtrA2/Omi shRNA–expressing retrovirus. (G and H) NIH3T3 cells infected with Ha-RasV12–expressing retrovirus were treated with 50 µM Ucf-101 for 2 h. (N and O) NIH3T3 cells were infected with a control or Ha-RasV12–expressing retrovirus and a control or HtrA2/Omi shRNA–expressing retrovirus. (P–R) NIH3T3 cells were infected with Ha-RasV12– and HtrA2/Omi shRNA–expressing retroviruses together with a control or p130Cas shRNA–expressing retrovirus. (A, C, F, and H) The cleavage of β-actin was evaluated by immunoblotting. Black arrowheads indicate full-length β-actin, and white arrowheads indicate cleaved fragments. (B and D) The amount of cleaved fragments of β-actin relative to the total amount of β-actin was evaluated. Data represent the mean ± SD; n = 4; *, P < 0.01. (E and G) The levels of phosphorylated p130Cas (p-p130Cas; E and G) and HtrA2/Omi (E) were evaluated by immunoblotting. Blots of phosphorylated p130Cas and p130Cas were quantified and the relative values of p130Cas phosphorylation are shown. (I) Cells were stained for F-actin (middle) and G-actin (bottom). Ratio images of F/G-actin (top). Bar, 20 µm. (J and K) F/G-actin ratio (J) or F-actin (K) values were normalized with the mean value of the control cells. Data represent the mean ± SD of more than 30 cells. *, P < 0.01. (L and M) Confocal images of cells stained for F-actin (L) or for paxillin and phosphorylated p130Cas (M). Enlarged images of top panel were shown in bottom panel. Arrowheads indicate lamellipodia. Bars, 20 µm. (N and Q) Inverted invasion assays were performed. Images of serial Z-sections (10-µm intervals). Bars, 100 µm. (O and R) Cells invaded 30 µm or more were quantified. Data represent the mean ± SD; n = 3. *, P < 0.01. (P) The expression level of p130Cas was evaluated by immunoblotting.

Knockdown of HtrA2/Omi attenuates the cleavage of β-actin and restores p130Cas phosphorylation in Ras-transformed cells. (A and B) NIH3T3 cells, iMEFs, and p53−/− MEFs were infected with a control or Ha-RasV12–expressing retrovirus. (C and D) NIH3T3 cells were infected with a Ha-RasV12–expressing retrovirus together with a control or p53R175H-expressing retrovirus. (E) NIH3T3 cells were infected with a control or Ha-RasV12–expressing retrovirus together with a control or HtrA2/Omi shRNA–expressing retrovirus. (F, and I–M) NIH3T3 cells were infected Ha-RasV12–expressing retrovirus and a control or HtrA2/Omi shRNA–expressing retrovirus. (G and H) NIH3T3 cells infected with Ha-RasV12–expressing retrovirus were treated with 50 µM Ucf-101 for 2 h. (N and O) NIH3T3 cells were infected with a control or Ha-RasV12–expressing retrovirus and a control or HtrA2/Omi shRNA–expressing retrovirus. (P–R) NIH3T3 cells were infected with Ha-RasV12– and HtrA2/Omi shRNA–expressing retroviruses together with a control or p130Cas shRNA–expressing retrovirus. (A, C, F, and H) The cleavage of β-actin was evaluated by immunoblotting. Black arrowheads indicate full-length β-actin, and white arrowheads indicate cleaved fragments. (B and D) The amount of cleaved fragments of β-actin relative to the total amount of β-actin was evaluated. Data represent the mean ± SD; n = 4; *, P < 0.01. (E and G) The levels of phosphorylated p130Cas (p-p130Cas; E and G) and HtrA2/Omi (E) were evaluated by immunoblotting. Blots of phosphorylated p130Cas and p130Cas were quantified and the relative values of p130Cas phosphorylation are shown. (I) Cells were stained for F-actin (middle) and G-actin (bottom). Ratio images of F/G-actin (top). Bar, 20 µm. (J and K) F/G-actin ratio (J) or F-actin (K) values were normalized with the mean value of the control cells. Data represent the mean ± SD of more than 30 cells. *, P < 0.01. (L and M) Confocal images of cells stained for F-actin (L) or for paxillin and phosphorylated p130Cas (M). Enlarged images of top panel were shown in bottom panel. Arrowheads indicate lamellipodia. Bars, 20 µm. (N and Q) Inverted invasion assays were performed. Images of serial Z-sections (10-µm intervals). Bars, 100 µm. (O and R) Cells invaded 30 µm or more were quantified. Data represent the mean ± SD; n = 3. *, P < 0.01. (P) The expression level of p130Cas was evaluated by immunoblotting.

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