Figure 3. Effect of EBP50 on vascular smooth muscle cell cycle and p21^cip1 expression.

A. Cell cycle distribution analyzed by FACS in WT (gray bars) and KO (white bars) VSMC. The percentage ± standard error of the population of cells in G1, S and G2/M phases are derived from five independent experiments. P values for WT vs. KO are indicated. B. Immunoblots for p21^cip1, p27^kip1 and p53 in WT and KO VSMC. β-actin was used as loading control. Blots are representative of at least five independent experiments. C. Quantification of the expression of p21^cip1 in WT (gray bars) and KO (white bars) VSMC. The ratio of p21^cip1 expression over β-actin was determined in five independent experiments. *, p<0.04. D.Quantitative RT-PCR for p21^cip1 and p27^kip1 using mRNA from WT (gray bar) and KO (white bar) VSMC. Results are presented as fold over WT ± standard error of triplicate determinations. Values were normalized relative to GAPDH. E. Inhibition of p21^cip1 expression by siRNA increases VSMC proliferation. Immunoblot for p21^cip1 in KO VSMC transfected with either control siRNA (si-C) or p21^cip1siRNA (si-p21^cip1) as indicated. β-actin is used as loading control. Proliferation of KO VSMC transfectedwith either control siRNA (si-C) or p21^cip1 siRNA (si-p21^cip1) and maintained in 10% FBS determined by [³H]-thymidine incorporation. Data are presented as percentage of si-C treated cells ± standard error of triplicate determinations. Similar results were obtained in two additional experiments. *, p<0.003.