Biologic function of the FGF2/LYVE-1 interaction in the endothelial cells. FGF2 stimulated migration (A), invasion (B), and proliferation (C) of LECs in the presence of soluble LYVE-1 (250 ng/mL) and/or FGF2 (20 ng/mL). Cells were incubated at 37°C 5% CO₂ in the IncuCyte live-cell imaging system as described in “Methods.” The time course of cell migration or invasion was quantified by dynamic imaging (pictures at 2-hour time intervals) as percentage of scar recovery (cells migrated/invaded into the wound). The proliferation was quantified by dynamic imaging using percentage of confluence at 2 hour time intervals. In the insets, diagrams of 1 time point are represented. Experiment was performed 3 times and 1 representative experiment is shown (n = 4 for proliferation; n = 6 for migration and invasion; *P < .05,** < .01,***P < .001; NS, nonsignificant versus control cells stimulated by FGF2 in the absence of LYVE-1). (D) Effect of soluble LYVE-1 on ERK phosphorylation using AlphaScreen SureFire phosphorylation assay. LECs were stimulated with 20 ng/mL FGF2 in the presence or absence of 250 ng/mL LYVE-1 for 5, 10, and 15 minutes. Phosphorylated ERK1/2 in cell lysates was quantified in an AlphaScreen SureFire p-ERK1/2 (Thr202/Tyr204) Assay (PerkinElmer) according to the manufacturer's instructions. Results are representative experiments from 3 independent experiments. Results are mean values ± SD (n = 3).