RhoB regulates cell area recovery after acute contraction independently of cell–cell contacts. (A) HA-RhoBV14 has no effect on endothelial cell–cell junctions. HA-RhoBV14 was expressed for 48 h in confluent HUVECs. Bottom images show enlargements of squared areas from HA-RhoBV14 transfected (1) and untransfected cells (2). Bar, 20 µm. (B) Time-lapse microscopy of subconfluent HUVECs stimulated with thrombin. Cell areas were quantified from individual frames taken at different times (right graph). Graph shows the mean + SEM of at least 10 cells per experiment from three different experiments. Bar, 20 µm. (C) siRNA-transfected, TNF-pretreated HUVECs were stimulated with thrombin for the indicated times, and then fixed and stained for F-actin and RhoB. Right graph quantifies cell area. Mean + SEM from three different experiments. 50 cells per experiment. RhoB-depleted cells recover their initial area (respreading) at 60 min poststimulation. *, P < 0.04; **, P < 0.03. (D) HUVECs expressing GFP or HA-RhoBV14 for 24 h were stimulated with thrombin for 60 min, stained for F-actin and HA-RhoBV14, and the cell area quantified. Graph shows the mean + SEM of at least 10 cells per experiment from three different experiments. **, P = 0.001. (E) siRNA-transfected, TNF-pretreated HDMVECs were stimulated with thrombin for 2.5 h (respreading), and then fixed and stained for F-actin and RhoB. Graph shows the mean + SEM from at least 50 cells per experiment, from three different experiments. *, P ≤ 0.05; **, P < 0.01. Bar, 10 µm.