Contribution of VEGFR-2 and NRP-1 to endothelial cell tube formation and migration. (A) HUVECs (60 × 10⁴) obtained 48 hours after transfection were suspended in HUVEC medium, plated on matrigel-coated 24-well plates, and incubated for 24 hours. Images reflect representative fields visualized by phasecontrast microscopy (original magnification, 10 ×). Results were reproduced in 5 different experiments. (B) Quantitative analysis of matrigel-induced tube formation in control and NRP-1–defective HUVECs. Tube formation under the conditions described in “VEGFR-2 and NRP-1 regulate migration and tube formation in HUVECs” was measured as a function of the number of vascular joints per visual field (10 × magnification) and expressed as an average of 4 nonoverlapping fields. The results represent the average (± SD) from 4 independent experiments. (C) Contribution of VEGFR-2 and NRP-1 to VEGF-induced endothelial cell migration. HUVEC transfected 24 hours earlier with control (black bars), VEGFR-2 (red bars), or NRP-1 siRNA (cream bars) were placed in the upper chamber of transwells precoated with gelatin; VEGF (100 ng/mL) was placed in the lower chamber. The results reflect the mean (± SD) number of migrated cells from 3 independent experiments.