Figure I. Retinoids Block the Progression of Human CASMC Into S-Phase

Figure I. Retinoids Block the Progression of human CASMC into S-phase

Cells were treated as described in Figure 1, except that retinoids were added at 2 mmol/L. Twenty-four hours after mitogenic stimulation, DNA was stained with propidium iodide (PI) and 1x106 cells were analyzed by flow cytometry. Figures represent DNA histograms for quiescent human CASMC (0.4% FBS for 24 h, 2A); CASMC stimulated with PDGF and insulin (P+I) alone (B); CASMC stimulated in the presence of TTNPB (C), atRA (D), AGN4204 (E), and 9cRA (F), respectively. The x and y axes represent the intensity of PI fluorescence and cell number, respectively. Data are representative of three separate experiments.

Figure II. Quantification of the G1 ® S block induced by retinoids at the highest concentration (2 mM)

Results are presented as mean ± SEM for percentage of cells progressing into S phase as measured by FACS analysis 24 h after quiescent human CASMC were stimulated with PDGF + insulin (S-phase transition), n=3, *p<0.05, **p<0.01 versus mitogen-stimulated cells.

Figure III. Retinoids inhibit serum-induced Rb phosphorylation in human CASMC

Quiescent human CASMC were preincubated with 2 mmol/L of each retinoid for 30 min prior to addition of 10% serum. After 24 h, whole cell proteins (35 mg) were assayed by Western immunoblotting using anti-phospho Rb Ser 807/811 antibody. Each blot is representative of three separate experiments.

Figure IV. Effect of retinoids on mitogen-induced expression of cyclins D1 in human CASMC

Cells were treated as described in Figure 1. After 24 h, whole cell protein (35 mg) were assayed by Western immunoblotting using anti-cyclin D1 antibody (upper panel). Each blot is representative of three separate experiments. Densitometric analysis of immunoblots is shown as % of mitogen-stimulated cells (lower panel). Results are presented as mean ± SEM (n=3), *p<0.05, **p<0.01 versus mitogen-stimulated cells.

Figure V. Retinoids inhibit mitogen-induced expression of cyclin A in human CASMC

Cells were treated as described in Figure 1. After 24 h, whole cell protein (35 mg) were assayed by Western immunoblotting using anti-cyclin A antibody (upper panel). Each blot is representative of three separate experiments. Densitometric analysis of immunoblots is shown as % of mitogen-stimulated cells (lower panel). Results are presented as mean ± SEM (n=3), *p<0.05, **p<0.01 versus mitogen-stimulated cells.