VEGF up-regulates Gas1 expression in HUVECs. (A) Quantitative RT-PCR analysis of Gas1 mRNA expression. Cells were seeded in sparse (4 × 10³ cells/cm²) or confluent (5 × 10⁴ cells/cm²) conditions in complete medium. Before addition of VEGF (80 ng/mL for 24 hours) cells were starved for 24 hours in serum-free medium supplemented with 1% BSA. Confluent cells were added with 50 μg/mL of anti–VE-cadherin–blocking mAb (BV9) for 1 hour before addition of VEGF. Data are means ± SD of 3 separate experiments performed in triplicate and are expressed as relative amounts using “sparse, VEGF -, mAb anti–VE-cadherin -” cell lysate as reference value. (B) Western blot analysis of Gas1 expression after VEGF treatment. (C) Time course of Gas1 mRNA expression after VEGF treatment. Confluent cells were activated with VEGF for the indicated times as described above. Data are means of 2 experiments performed in quadruplicate.