Stimulation with ephrin B1-Fc or ephrin B2-Fc enhances SDF-1-induced HUVEC chemotaxis. (A) HUVECs (0.5 × 10⁶) preincubated (10 minutes, 37°C) in medium only, with ephrin B1-Fc (1 μg/mL), or with ephrin B2-Fc (1 μg/mL) were placed on the top chamber of the transwell. SDF-1α (50 ng/mL) or chemotaxis medium only was placed in the bottom chamber. (B) HUVECs (0.5 × 10⁶) were first incubated (20 minutes) in medium only, with the peptide SNEW (100 μM), or with a control scrambled form of SNEW (SCR-EPQ, 100 μM). Cells were subsequently incubated (10 minutes, 37°C) with medium only or with ephrin B1-Fc (1 μg/mL). Cells and additives were placed on the top chamber of the transwell. SDF-1α (50 ng/mL), or chemotaxis medium only was placed in the bottom chamber, and the transwells were incubated for 18 hours at 37°C. (C) HUVECs (0.5 × 10⁶) were first incubated (20 minutes, 37°C) in medium only, with the peptide TNYL-RAW (100 μM), or with the peptide SNEW (100 μM); cells were subsequently incubated (10 minutes, 37°C) with medium only or with ephrin B2-Fc (1 μg/mL). Cells and additives were placed on the top chamber of the transwell. SDF-1α (50 ng/mL) or chemotaxis medium only was placed in the bottom chamber. All transwell plates (A-C) were incubated 18 hours at 37°C. Cells accumulated into the lower chamber were counted. Results reflect the mean ± SD number of migrated HUVECs under the different conditions tested (3-5 separate experiments, each performed in triplicate).