Silencing of Rap1 inhibits endothelial migration. (A) HUVECs were transfected with GFP-RBD_RalGDS. Six hours after transfection, a wound was created. Representative images of 3 different experiments are depicted, showing recruitment of the GFP-RBD_RalGDS (as an indicator of Rap1 activity) at the leading edge of migrating endothelial cells. Micrographs were acquired with an LSM 510 confocal microscope fitted with a Plan-Neofluar 40×/1.3 numeric aperture (NA) oil objective and LSM5 image acquisition software (all from Carl Zeiss). (B) HUVECs were transfected with Rap1GAP1-FLAG or empty vector. After 24 hours, cells were seeded in the upper chamber of modified Boyden chambers coated with fibronectin (n = 5) or collagen (n = 5). Endothelial cell migration was stimulated using VEGF (50 ng/mL) as chemoattractant where indicated. Data are presented as mean of migrated cell % of control plus or minus SEM (*P < .05 vs empty vector, **P < .05 vs empty vector + VEGF). (C) Migration assay on fibronectin (n = 5) with endothelial cells transfected with siRNAs targeted against Rap1a, Rap1b Rap1a/Rap1b, or scrambled siRNA. After 48 hours, cells were seeded in the upper chamber of modified Boyden chambers. Endothelial cell migration was assessed using VEGF (50 ng/mL) as chemoattractant. Data are presented as mean of migrated cell % of control plus or minus SEM (*P < .05 vs scrambled siRNA, #P < .05 vs scrambled siRNA + VEGF).