Figure 6

Involvement of GEF-H1 in vincristine-enhanced invasive ability. A. At 72 or 92 h after transfection of GEF-H1-specific siRNA or negative control siRNA, cells were harvested to analyze GEF-H1 expression by Western blotting using anti-GEF-H1 and anti-actin antibodies. Actin was used as a loading control. B. At 72 h after transfection, starved cells (24 h) were treated with or without 15 μM vincristine for 15 min. The cells were then lysed and analyzed by Western blotting. The blots were quantified by densitometry, and the results were expressed as a ratio relative to the values of pMLC/MLC obtained in non-treated cells. The graph shows mean ± S.E. of three independent experiments. **, P < 0.01. C. Control siRNA- or GEF-H1 siRNA-transfected cells on gelatin-coated coverslips were treated with vehicle or 15 μM vincristine. The cells were fixed and stained with Alexa Fluor 488 phalloidin and DAPI. F-actin (green) and nuclei (blue) were analyzed using confocal microscopy. Arrows indicate the cells with membrane blebs. White bars, 10 μm. The graph shows means ± S.E. of three independent experiments. **, P < 0.01. D. The number of invading cells measured by invasion assay. At 72 h after transfection, cells (1 × 10⁶) were seeded into the upper chamber with or without 15 μM vincristine. After 24 h incubation, the invading cells were fixed, and stained with toluidine blue. Total numbers of the stained cells were counted using a microscope. The graph shows means ± S.E. of three independent experiments. *, P < 0.05.