SEMA3F inhibits HMVEC survival induced by VEGF-A and VEGF-C. (A) Supernatants from HEK cells transfected with empty vector or SEMA3F were analyzed by Western blot analysis. Twenty-four hours after transfection, supernatants were harvested and replaced with serum-free media for additional 24 hours. Supernatants were then collected, centrifuged, and filtered prior to concentration. An aliquot containing 20 μg total proteins was analyzed by Western blot. (B-C) Survival assay of HMVECs stimulated in the presence of control or SEMA3F-concentrated medium. Cells were seeded in triplicate in gelatinized 96-well plates in medium containing 0.1% FCS. Cells were then left unstimulated or stimulated with VEGF-A 10 ng/mL (B) or VEGF-C 100 and 300 ng/mL (C) in the presence of control- or SEMA3F-conditioned media (***P < .001 versus the respective stimulation of cells incubated with the control medium). Data are shown as mean ± SEM. Results are representative of 2 independent experiments.