FIG. 9.
Determination of the role of RhoA/ROCK signaling in regulation of Cip/Kip CDKIs. (A) Effect of RhoA knockdown on expression of cell cycle regulators. siRNA against RhoA (10 nM) was applied to control cells during the serum starvation step of the synchronization procedure. Following synchronization, cells were released for the time periods indicated. Western blotting was undertaken as indicated. Data shown are representative of at least three independent experiments. (B) Effect of RhoA knockdown on cell cycle progression. Entry into S phase after a 6-h release was determined by uptake of BrdU during a 2-h pulse. The incorporation of BrdU was measured by flow cytometry. Data shown represent the mean ± standard error of three independent experiments. *, P < 0.05. (C) Effect of ROCK inhibition on expression of p21WAF1/Cip1 and p27Kip1. The ROCK inhibitor Y27632 was included during the hydroxyurea (HU) treatment and during release in growth medium at the concentrations indicated. Cell lysates were subjected to Western blotting as indicated. Data shown are representative of at least three independent experiments. (D) Effect of ROCK inhibition on cell cycle progression. Cells were treated as described for panel C, and entry into S phase after a 6-h release was determined by uptake of BrdU during a 2-h pulse. The incorporation of BrdU was measured by flow cytometry. Data shown represent the mean ± standard error of three independent experiments. *, P < 0.05.