Ec growth supplement

EC isolation was performed as described previously with modifications ^{70 (link)}. Briefly, E10.5 Snail1^fl/fl embryos (of either male or female sex) or adult lungs (isolated from female mice) were dissected in ice-cold PBS and digested in a mixture of collagenase type I (Worthington), DNase I (Sigma-Aldrich) and dispase (Invitrogen) for 30 min at 37°C. ECs were then separated using Dynabeads (Invitrogen) coated with anti-PECAM-1 antibody and cultured in DMEM medium supplemented with 20% fetal bovine serum (FBS, Invitrogen) and EC growth supplement (BD Biosciences). Confluent ECs were trypsinized and separated using Dynabeads coated with anti-ICAM-1 antibody (BD Biosciences). Following two rounds of sorting, the purity of ECs is ~90% and the cells were used within two passages of their initial isolation. To obtain Snail1 WT and KO ECs, cells were infected with adenoviral-βGal or adenoviral-Cre (MOI, 50), respectively. For in vitro assessments of EC morphogenesis, Snail1 WT and KO ECs were cultured atop Matrigel-coated dishes (BD Biosciences) in the absence or presence of DAPT (8 μm) for 12 h and imaged ^{4 (link)}. In selected experiments, ECs were freshly isolated from E10.5 WT and Snail1 LOF embryos using Dynabeads coated with anti-PECAM1 antibody, and subjected to microarray gene expression and qRT-PCR analyses.