Confluency and VE-cadherin up-regulate Gas1 in endothelial cells. (A) Quantitative RT-PCR analysis of Gas1 mRNA in freshly isolated HUVECs. Confluent (5 × 10⁴/cm²) and sparse (4 × 10³/cm²) cells were cultured for 24 hours in the presence or absence of 20% FCS. At the end of this period mRNA was extracted as described in “Materials and methods.” RT-PCR data are means ± SD of 3 separate experiments performed in triplicate and are expressed as relative amounts using “sparse cells/serum -” as reference value. (B) Quantitative RT-PCR analysis of isogenic endothelial cells VE-cadherin–null or expressing the VE-cadherin wild-type (VE-cadherin positive) or truncated (tVE) cDNA. Cells were seeded at the same density and cultured for 3 days in the presence of 10% FCS and then extracted for Gas1 mRNA evaluation. RT-PCR values are means ± SD of 4 different experiments performed in triplicate and are expressed as relative amounts using “VE-cadherin null” cell lysate as reference value.