Figure 10.

Reducing SGEF expression in HUVECs blocked RhoG activation and TEM. (A) siRNA against SGEF reduces the expression of endogenous SGEF in endothelial cells. The siRNA did not affect the expression levels of endogenous RhoG, Rac1, ICAM1, or moesin. (B) Knockdown of SGEF expression by siRNA inhibits the activation of RhoG downstream from ICAM1. HUVECs were transiently transduced with myc-RhoG-wt and treated with TNF-α. SGEF expression was reduced by siRNA in HUVECs, and RhoG activity, which was induced by αICAM1 beads, was measured using GST-ELMO. The top panel shows RhoG activity after 30 min, which was depressed when SGEF expression was reduced (bottom panel). The second panel shows equal expression for myc-RhoG-wt in HUVECs. The third panel shows equal levels of ICAM1 in cell lysates. The bottom panel shows reduced SGEF expression after siRNA treatment in HUVECs. The experiment was performed two times. (C) Knockdown of SGEF expression decreased ICAM1 cup formation. Endothelial cells transfected with SGEF siRNA were incubated with HL60 cells. Quantification of ICAM1-positive rings around adhered leukocytes, which was measured as described in Materials and methods, shows that cup formation was significantly decreased when SGEF levels were reduced. *, P < 0.05. (D) Knockdown of SGEF expression inhibited transmigration. Endothelial cells were cultured on transwell filters and transfected with the appropriate siRNA as described in Materials and methods. 48 h later, differentiated HL60 cells were allowed to transmigrate for 4 h under spontaneous conditions (black bars) or toward 50 ng/ml SDF-1 in the lower chamber (white bars). Reduced SGEF levels diminish SDF-1–induced transmigration significantly. *, P < 0.001; **, P < 0.05. (C and D) The experiment was repeated nine times. Data are means ± SEM (error bars).