Figure 6.

RND1 is required for Notch-mediated suppression of EC migration. (A) Lentivirally-transduced HUVECs with RFP or DLL4 expression constructs were transfected with siCNT or siRND1 and plated in modified Boyden chambers and incubated with 50 ng/ml of VEGF for 6 h. Cells that had migrated through the pores were counted and normalized to their respective controls. DLL4 overexpression activation of Notch signaling suppresses endothelial migration (third column), but knockdown of RND1 rescues the endothelial migration to nearly control levels. (B) Representative images of cells that had migrated through Boyden chamber pores under each condition in (A). (C) Control and siRND1 transfected HUVECs were plated on a Boyden chamber inserts coating with fibronectin (5 µg/ml) and either Fc (control) or 10 µg/ml DLL4-Fc (Notch activation) and incubated with 50 ng/ml of VEGF for 6 h. Physiologic activation of Notch signaling by tethered DLL4 ligand suppresses endothelial migration (third column), but knockdown of RND1 increases migration under endogenous Notch signaling conditions (second column) or ligand-induced Notch signaling (fourth column). (D) Representative images of cells that had migrated through Boyden chamber pores under each condition in (B). The scale bar indicates 210 μm.