Sema6A silencing decreases VEGFR2 levels in endothelial cells. (A) VEGFR2 levels in HUVECs 72 hours after infection with control or Sema6A shRNA. Representative flow cytometry profiles display VEGFR2 levels (hi indicates high; lo, low; and neg, negative) as a function of DNA content (DAPI staining after cell permeabilization); Ap indicates apoptotic cells. (B) Relative levels of Sema6A expression in HUVEC control (control shRNA) and Sema6A-silenced (Sema6A shRNA) attached or floating 72 hours after infection measured by quantitative PCR. (C) Levels of VEGFR2 and cleaved caspase3 in cell lysates of HUVECs 72 hours after infection with control or Sema6A shRNA detected by immunoblotting; β-actin was used as loading control. The Sema6A-silenced cells were examined as attached, floating and all cells. Normalized (VEGFR2/β-actin) band intensity values are displayed. After infection with control or Sema6A shRNAs and 7-day puromycin selection, HUVECs were tested for VEGFR2 expression by flow cytometry (D) and quantitative PCR (E). The results of PCR are expressed as mean gene expression (± SEM; n = 5) relative to control. (F) VEGFR2 protein was evaluated by immunoblotting in cell lysates of Sema6A-silenced and control HUVECs (after 7-day puromycin selection). (G) Relative levels of plexinA2 (PLXNA2), plexinA4 (PLXNA4), and VEGFR2 expression in HUVEC control (shRNA control) and in HUVECs subjected to shRNA knockdown of PLXNA2, PLXNA4, PLXNA2+PLXNA4, or VEGFR2. The results of PCR reflect the means RNA levels (± SD; n = 4) relative to control. (H) VEGFR2 levels in cell lysates of HUVEC control (shRNA control) and in HUVECs with shRNA knockdown of PLXNA2, PLXNA4, PLXNA2+PLXNA4, or Sema6A. Relative band intensities values (normalized to β-actin) are displayed. (I) Relative levels of VEGFR2 mRNA in attached control (shRNA control) and Sema6A-silenced (shRNA Sema6A) HUVECs supplemented with IgG1-Fc or recombinant Sema6A-Fc (0.4-10nM). VEGFR2-silenced (shRNA VEGFR2) HUVEC were used as a control (*P < .001).