Figure 3

RhoGDIβ expression in H9c2 cells retards cell cycle progression. (A) Growth rate of H9c2 cells stably transfected with pTet or pTet-RhoGDIβ grown in 10% fetal bovine serum containing doxycycline. (B) Analysis of RhoGDIβ-expressing cells by flow cytometry. RhoGDIβ-expressing H9c2 cells were grown in 10% fetal bovine serum with doxycycline for 3 days, and cells were stained with propidium iodide and subjected to flow cytometry. The percentages of cells in the different cell cycle phases were calculated using ModFit software. Experiments were performed three times and gave similar results. (C) Expression and activities of various regulatory cell cycle proteins in RhoGDIβ-expressing cells. Total cell extract was analyzed by western blotting and probing with the specific antibodies indicated. Cyclin D or E was immunoprecipitated from RhoGDIβ-expressing cells and analyzed for associated histone H1 kinase activity. JNK activity was analyzed by immunoprecipitating JNK and assessing its activity using GST-c-jun as a substrate.