CXCR4 expression and function in NRP-1–defective endothelial cells. (A) Expression of the integrins α₃, α₅β₁, and β₁ in HUVECs transfected 48 hours earlier with control (open black curve) or NRP-1 siRNA (open green curve) detected by flow cytometry. (B) Flow cytometric analysis of surface CXCR4 expression in HUVECs transfected 48 hours earlier with control, VEGFR-2, or NRP-1 siRNA. Filled red curves represent IgG2a (control); open black curves, CXCR4. (C) Representative confocal images showing the different distribution of intracellular CXCR4 in HUVECs transfected with NRP-1 or control siRNA. The cells were immunostained for CXCR4 (red) and counterstained with DAPI (blue). (D) CXCR4 internalization in NRP-1–silenced cells. HUVECs transfected with control or NRP-1 siRNA 48 hours earlier were incubated for 30 minutes at 37°C with the CXCR4 ligand SDF-1α (▦; 500 ng/mL) and then immunostained for surface CXCR4. ▪ indicates control medium only. Results reflect the mean (± SD) of 3 experiments performed. (E) SDF-1–induced ERK activation in HUVECs transfected with control or NRP-1 siRNA evaluated by Western blotting with specific antibodies to phosphorylated and total ERK. Cells obtained 48 hours after transfection were starved for 16 hours and then exposed for 15 minutes to 500 ng/mL SDF-1α. (F) Endothelial cell migration in response to SDF-1. HUVECs transfected 48 hours earlier with control or NRP-1 siRNA were placed in the upper chamber of transwells precoated with gelatin; SDF-1 (▦; 100 ng/mL) was placed in the lower chamber. ▪ indicates control medium only. The results reflect the mean (± SD) number of migrated cells from 4 independent experiments.