NFATc3 knockdown blocks COX2-dependent endothelial cell migration and proliferation. (A) Time course of HUVEC migration in wound-healing experiments. HUVEC monolayers in medium containing 1% serum were scratched, and VEGF (100 ng/mL) was added to stimulate endothelial cell migration. The migrated area was measured from captured images at the indicated times. The graphic shows 1 representative experiment out of 3 performed. (B) HUVECs expressing KDSc or KD-c3 were analyzed with an in vitro wound-healing assay. Shown is a photomicrograph of representative fields after 14 hours of stimulation. Dotted lines mark the limits of the unpopulated area. (C) Quantification of migrated area (the proportion of the denuded area repopulated by migrating cells) at 14 hours (mean ± SD; n = 3). (D) Wound-healing assay with VEGF-stimulated HUVECs expressing KDSc, KD-c3, or KD-c3 + COX2. Knockdown of NFATc3 and overexpression of human Cox2 were detected by Western blot (bottom). (E) Endothelial proliferation assay. HUVECs were incubated with VEGF (50 ng/mL) and ³H-thymidine (1μCi/mL) for 48 hours. Values of incorporated ³H-thymidine for each condition were normalized to the within-experiment mean. Data are the means ± SD of 3 independent experiments performed at least in triplicate. *P < .05, **P < .01, and ***P < .001 compared with control (KDSc).