RhoA is required for T cell crawling and TEM. (a) CEM T cell TEM on Transwells (n ≥ 5). (b) RhoA expression in CEM cells relative to GAPDH (n = 3). (c) Time of TEM completion determined by time-lapse microscopy (control siRNA, n = 32 cells; RhoA siRNA, n = 21 cells; four experiments). Boxes, median, 25th, and 75th percentiles; whiskers, 5th and 95th percentiles; dots, outliers. (d, left) Endothelial ICAM-1 localization around CEM cells on EC. Arrows, ICAM-1 clusters. ICAM-1 localizes around transmigratory channel (middle) or clusters around T cell processes of RhoA-depleted cells (bottom). (right) Quantification of ICAM-1 clustering around nontransmigrating cells and transmigratory path (n = 4; ≥100 cells/experiment). Insets: magnification of regions of ICAM-1 clustering indicated by blue arrows. (e, left) Tracks of 20 representative cells on EC from time-lapse videos. (right) Mean velocity of tracked cells (n = 4; ≥38 cells/experiment). (f) Images from time-lapse videos of CEM cells crawling on and transmigrating across EC. (right) Outlines of asterisk-marked control (yellow) and RhoA siRNA (blue and green) cells. Arrows, migration direction of control cells; dashed lines, transmigrated regions. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars indicate mean ± SEM. Bars, 20 µm.