Figure 4.

THs stimulate migration through PI3K/Rac activation in HUVECs. Before performing the experiment described below, HUVECs were incubated for 4 h at 37°C with RPMI 1640 medium containing 0.1% fatty acid–free BSA. A, HUVECs were pretreated with or without 100nM wortmannin or 1μM IOP for 20 min at 37°C. The cells were then stimulated for 10 min at 37°C with T₃ (1nM), T₄ (1nM), PBS (none), wortmannin, or IOP to measure the phosphorylated form and total Akt, activated Rac, and total Rac by Western blotting. B, HUVECs were infected with adenovirus carrying GFP or a dominant negative form of Rac1 before the experiment. The cells were then stimulated for 4 h at 37°C with T₃ (1nM), T₄ (1nM), or PBS (none) to investigate the migration of HUVECs using a blind Boyden chamber apparatus. The number of migrated cells attached to the lower surface of the filters was counted under a microscope at 400× magnification. C, HUVECs were incubated for 10 min at 37°C with PBS (none), rT₃ (1nM), T₃ (1nM), T₄ (1nM), or S1P (1μM) to analyze RhoA activation by Western blotting. Representative results of three separate experiments are shown in panels A and C. Results are presented as means ± SEM of three separate experiments in panel B. *, P < .05 vs none.