Endothelial cells require VEGF signaling for survival. (A) VEGFA gene expression in Sema6A-silenced and control HUVECs was measured by quantitative PCR. The results reflect the mean (± SEM; n = 4) relative mRNA levels. (B) HUVECs were incubated (2 hours) with Sorefanib at the indicated concentrations and then treated with VEGF-A (100 ng/mL; 15 minutes). Cells lysates were tested for VEGFR2 and ERK phosphorylation by immunoblotting. (C) Cell death was measured by flow cytometry in HUVECs incubated (18 hours) with medium only or medium supplemented with Sorafenib (10μM). (D) HUVECs were infected with control (shRNA control) or VEGFR2 silencing lentivirus (shRNA VEGFR2). Cell lysates were evaluated for VEGFR2 and cleaved caspase 3 by Western blotting. (E) Cell death in VEGFR2-silenced and control HUVECs was measured by flow cytometry (representative results). (F) The bar graph shows the percentage viable, apoptotic and necrotic VEGFR2-silenced and control HUVECs from 4 independent experiments (± SEM; P values reflect statistical significance of group differences). (G) VEGFA gene expression in VEGFA-silenced (VEGFA shRNA 343 or 345) and control HUVEC measured by quantitative PCR. (H) Control (shRNA control), Sema6A-silenced (shRNA Sema6A) or VEGFA-silenced (shRNA VEGFA) HUVEC were cultured for 6 hours in EBM-2 medium supplemented with FGF2, EGF, and IGF-1 from EGM-2 BulletKit. Avastin (A; lanes 4, 10, and 16) was added at 10 μg/mL; Sorafenib (S; lanes 5, 11, and 17) was added at 2μM; and Na₃VO₄ (lanes 3-5, 9-11, 15-17) was added at 100μM. Cells were stimulated with exogenous VEGF-A (100 ng/mL) for 5 minutes. Cell lysates were immunoblotted for phosphorylated VEGFR2 (tyrosine 1175 or 951), total VEGFR2 and β-actin.