sNpn-1 and sVEGFR-1 counteract VEGF₁₆₅ effects on Npn-1 expression and Sema3A function. (A) Binding of VEGF₁₆₅ (25 ng/mL) to Npn-1 (2 μg/mL)–coated wells with srNpn-1 (0-3.2 μg/mL) or sVEGFR-1 (0-3.2 μg/mL). Error bars depict ± SD of triplicate determinations. (B) Npn-1 on endothelial cells incubated (25°C, 1 hour) with or without srNpn-1 (5 μg/mL) (top left); VEGF₁₆₅ (25 ng/mL) plus srNpn-1 (0-800 ng/mL) (bottom left); VEGF₁₆₅ (25 ng/mL) plus control B7-1/Fc (0 or 800 ng/mL) (top right); and VEGF₁₆₅ (25 ng/mL) plus sVEGFR-1 (0-800 ng/mL) (bottom right). Dotted line: Npn-1 without stimulation of VEGF₁₆₅. (C) Npn-1 detected by confocal microscopy (scale bar, 20 μm) in cells incubated (37°C; 30 minutes) in medium only (left panel), with VEGF₁₆₅ (25 ng/mL), with VEGF₁₆₅ plus srNpn-1 (3.2 μg/mL), or with VEGF₁₆₅ plus sVEGFR-1 (3.2 μg/mL) (right panel). Images were acquired and processed as described for Figure 5F. (D) Lamellipodia retraction in endothelial cells incubated (37°C, 1 hour) with Sema3A (2 μg/mL), Sema3A (2 μg/mL) plus VEGF₁₆₅ (25 ng/mL), with or without sVEGFR-1 (0.8 or 3.2 μg/mL). Error bars depict ± SD of triplicate determinations. *P < .05; **P < .01.