PI3-kinase activation is required for Gas1 expression but not for its antiapoptotic activity. (A) Confluent HUVECs were treated with 0.1 μM Wortmannin (WM) 15 minutes before stimulation with 80 ng/mL VEGF for 24 hours. Gas1 synthesis was evaluated by Western blot. VEGF increases 3 times the amount of Gas1 in endothelial cell extracts (OD values: 5307 for resting cells and 14 879 for cells activated with VEGF); WM decreased both the basal and VEGF-induced Gas1 synthesis by 23% and 40%, respectively (OD values: 4126 vs 5307 for resting cells treated with WM and 8964 vs 14 879 for cells treated with VEGF and WM). Three independent experiments gave comparable results. (B) Cells were analyzed for Gas1 mRNA expression and cell apoptosis (C) by quantitative RT-PCR and DNA fragmentation, respectively. VEGF reduced apoptosis and increased Gas1 mRNA synthesis. Both effects were strongly inhibited by cell treatment with WM. The values are means ± SD of 4 separate experiments performed in duplicate. RT-PCR data are expressed as relative amounts taking “control cells” cell lysate as reference value. (D) Effect of WM on apoptosis of VE-cadherin–null and –positive cells transfected or not transfected with Gas1 cDNA. WM (0.1 μM) was added to the cells for the last 24 hours of duration of the experiment of apoptosis (Figure 4 legend). Data are means ± SD of 3 separate experiments performed in triplicate.