Rac1 activity is regulated by RhoB and is required for endothelial barrier recovery after acute contraction. (A and B) siRNA-transfected HUVECs were pretreated with TNF and stimulated with thrombin for the indicated times. Cells were lysed and pull-down assays performed to detect (active) Rac1-GTP levels. (A) Effect of RhoB siRNAs on Rac1 activation. Bottom graph shows active Rac1 normalized to total Rac1 levels. Mean + SEM from al least three different experiments measuring. *, P ≤ 0.05. (B) Effect of RhoA siRNA on Rac1 activation. Right graphs show the mean + SEM from three different experiments measuring the absolute levels of Rac1-GTP (active) and total Rac1. ***, P = 8.85 × 10−6. (C and D) Rac inhibition impairs barrier recovery in RhoB-transfected cells. siRNA transfected, TNF-pretreated HUVECs were stimulated with thrombin. 20 min after stimulation, 100 µM Rac inhibitor NSC23766 was added when indicated. (C) Cells were fixed and stained for the indicated proteins 72 min after stimulation. Automated image analysis was applied to detect empty areas in the cell monolayer (mask). Bar, 20 µm. (D) Analysis of TEER at 30 and 72 min after stimulation. Graph shows the mean + SEM of five different experiments. *, P ≤ 0.036.
