Figure 5.

The effect of genistein on thrombin-induced endothelial permeability is mediated by inhibition of RhoA[b]. A, BAEC monolayers were incubated for 24 h with 5 μg/ml C3 transferase (C3), heat-inactivated C3 (IC3), or vehicle (C), followed by addition of 2 U/ml thrombin (T) for 1 hour to stimulate and measure avidin-FITC passage. B, BAECs were preincubated with 5 μg/ml C3 transferase (C3), inactivated C3 (IC3), or vehicle (C) for 24 hours. Cells were then stimulated with thrombin (T; 2 U/ml) for 5 minutes. Phosphorylation of MLC (P-MLC) and total MLC were detected by Western blot. Representative images from three independent experiments with similar results are shown. C–E, BAECs were preincubated with 5 μg/ml C3 transferase (C3), inactivated C3 (IC3), or vehicle (C) for 24 hours. The cells were then incubated with genistein (G, 0.1–10 μM) for 30 minutes prior to addition of thrombin (T; 2 U/ml) for 5 minutes. The plasma membranes (PM) and cytoplasm (Cyto) of the cells were isolated, and RhoA in the PM, cytosol, and whole cell extracts (Total) were detected by Western blots. F, BAEC monolayers were incubated with 5 μg/ml C3 transferase (C3) or vehicle (C) for 24 hours, followed by the addition of 5 μM genistein (G) for 30 minutes before stimulation with 2 U/ml thrombin (T) for 1 hour. Avidin-FITC passage was measured. All data are expressed as mean ± SE of four independent experiments, each performed in duplicate.*P < .05 vs control; †P < .05 vs thrombin-alone-treated cells; #P < .05 vs ≤5 μM genistein-treated cells (D) or ≤ 1 μM genistein-treated cells (E).