NRP1 is dispensable for cell matrix adhesion but promotes FN-induced cell spreading, filopodia extension, and motility of primary ECs. (A–C) Immunoblotting with the indicated antibodies to demonstrate NRP1 knockdown (A) and time course of cell adhesion (B and C) in HDMECs and HUVECs transfected with control or NRP1 siRNA and plated for the indicated times on plastic dishes coated with 10 µg/ml FN (mean ± SEM of 3 independent experiments). (D–H) HDMECs transfected with control, VEGFR2 or NRP1 siRNA were plated on FN for the indicated times before immunoblotting (D), qPCR analysis (E) or fluorescent labeling (F) with the F-actin marker phalloidin (green) and the nuclear counterstain DAPI (blue). Bar, 20 µm. Images shown in the right two columns are higher magnifications of areas indicated with dotted squares. Also shown is a quantification of cell area (G) and phalloidin-stained microspikes (H) at the indicated time points (mean ± SEM of ≥30 cells from 3 independent experiments). (I) HDMECs transfected with si-NRP1 were plated on FN in the presence of 50 ng/ml VEGF165 for the indicated times before fluorescent labeling with phalloidin (green) and DAPI (blue). Bar, 20 µm. (J and K) HDMECs transfected with control or NRP1 siRNAs were plated on FN and cell motility on FN observed for 200 min. J shows representative tracks, with the point of origin of each cell plotted as 0 at the axis intersection. K shows mean track length (mean ± SEM of 23 cells from 2 independent experiments). (L–N) HDMECs or HUVECs transfected with control, VEGFR2 or NRP1 siRNA (L) and Nrp1^fl/fl MLECs transfected with adenovirus carrying Gfp control or Cre transgenes (M and N) were plated on FN-coated transwells. The number of transmigrated cells was determined after 240 min (mean ± SEM from ≥3 experiments, each performed in duplicate). Asterisks indicate statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test.