Thrombin increases RhoB vesicular trafficking and reduces vesicular RhoB nanoclusters during cell area recovery. (A–C) GFP-RhoB was expressed in HUVECs for 24 h. TNF-pretreated cells were left unstimulated (−) or stimulated with thrombin for at least 60 min and analyzed by time-lapse confocal microscopy at 20-s intervals during a 20-min period. Bars: 20 µm; (zoom) 2 µm. (B) RhoB-positive vesicles emerging from a perinuclear compartment toward the plasma membrane were tracked and the tracks plotted with a common origin. 12 cells per condition were analyzed. Bar, 2 µm. (C) RhoB-positive vesicles emerging from the plasma membrane toward a perinuclear compartment were tracked and the tracks plotted with a common origin. 12 cells per condition were analyzed. Bar, 2 µm. (D) STORM microscopy to generate a superresolution image of RhoB in perinuclear vesicles in TNF-pretreated HUVECs. Bar, 2 µm. (E) STORM images of RhoB clusters in intracellular vesicles during thrombin stimulation in TNF-pretreated HUVECs. Pixels of the reconstruction are shown. RhoB clustering was measured by calculating the Ripley´s K-function. K function for clusters from at least five images in three different experiments was calculated. ***, P < 0.005. Bar, 2 µm. (F) Localization of endogenous RhoB in TNF-pretreated HUVECs previously incubated with transferrin-TRITC for 5 min to localize early endosomes and for 1.2 h to localize recycling endosomes during barrier recovery. Localization of endogenous RhoB upon expression of the indicated Rab-GFP proteins. RhoB clearly colocalizes with Rab7-positive vesicles (arrowhead) in a perinuclear compartment (bracket).