Expression of the phosphomimetic form of HtrA2/Omi enhances β-actin cleavage and suppresses Ras-driven cell invasion. (A) Ras-transformed p53R175H-expressing cells were transfected with a Flag-tagged wild-type (WT) or S142D mature HtrA2/Omi (mHtrA2) expression vector. 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 evaluated. Anti-Flag and anti-HtrA2/Omi antibodies were used to examine the expression level of exogenous HtrA2/Omi. Black arrows indicate exogenous HtrA2/Omi, and white arrow indicates endogenous HtrA2/Omi. N.S., nonspecific band. (B) Purified proteins of wild-type (WT), S306A, or S142D mature HtrA2/Omi were subjected to SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining or anti-Flag immunoblotting. (C) Purified HtrA2/Omi proteins in B were incubated with F-actin for indicated time. Images were obtained by TIRF microscopy. Bar, 5 µm. (D) Serial time-lapse images of the actin filament in the red rectangle in C. Numbers indicate the time in seconds. Bar, 1 µm. (E) Fluorescence intensities after incubation with each form of purified HtrA2/Omi relative to fluorescence intensities before the incubation were calculated. Data represent the mean ± SD; n = 3. *, P < 0.05. (F and G) Ras-transformed p53^−/− MEFs were transfected with a control or Flag-tagged S142D mature HtrA2/Omi (mHtrA2) expression vector. (F) Inverted invasion assays were performed. Images of serial Z-sections (10-µm intervals). Bars, 100 µm. (G) Cells that had invaded 30 µm or more were quantified. Data represent the mean ± SD; n = 3. *, P < 0.01.