Involvement of ROCK in vincristine-enhanced invasive ability. A. Cells were starved in serum-free RPMI1640 for 24 h, and then treated with or without 15 μM vincristine and/or 50 μM Y27632 for 15 min. The cells were then harvested to evaluate MLC phosphorylation by Western blotting using anti-MLC and anti-pMLC antibodies. The blots in (A) are representative of four independent experiments. The blots were quantified by densitometry, and the results were expressed as ratio relative to the values obtained in non-treated control cells. The graph in (A) shows mean ± S.E. of four independent experiments. *, P < 0.05; **, P < 0.01. B. Cells on gelatin-coated coverslips were treated with drugs, 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 < 0.05. C. Cells (1 × 106) were seeded into the upper chamber with or without 15 μM vincristine and/or 50 μM Y27632. 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 graphs show means ± S.E. of three independent experiments. *, P < 0.05.