Silencing Sema6A expression promotes endothelial cell death. (A) HUVECs were infected with lentivirus control (shRNA control) or Sema6A-silencing lentivirus (shRNA Sema6A); Sema6A expression was measured by quantitative PCR after 7-day puromycin selection. The results reflect the means (± SD) of 4 experiments. (B) Seventy-two hours after infection, cells were stained with propidium iodide and Hoechst 33342, and the distribution of viable (V), necrotic (N), and apoptotic (Ap) cells was measured by flow cytometry. (C) Growth kinetics of HUVEC measured by IncuCyte monitoring over 96 hours culture, beginning 72 hours postinfection with control or Sema6A shRNA. The results are expressed as mean (± SEM, 4 replicate wells) percentage culture confluence/time point. (D) Cell death was measured by flow cytometry in Sema6A-silenced and control HUVEC (after lentivirus infection and 7-day puromycin selection). Results are from 4 independent experiments (± SEM). (E) Cleaved caspase3 in Sema6A-silenced and control HUVEC detected by immunofluorescence staining. (F) Sema6A-silenced and control HUVEC were cultured in complete culture medium alone or with VEGF (VEGF-A 100 ng/mL) or FGF (FGF2 100 ng/mL). Results from IncuCyte monitoring are expressed as mean (± SD; 4 replicate cultures) percentage culture confluence (48 hour time point). P = statistical significance of group differences.