Both N- and VE-cadherin decrease FoxO1 and β-catenin transcriptional activity. (A-B) Immunoblots and quantification of phospho (p)–AKT, total AKT, p-FoxO1, total FoxO1 from N, VE and N+VE cells. Columns are means± SEM, (n = 3; *P < .05, **P < .02). (C) FoxO1 luciferase activity. Results represent the means ± SD of relative light units determined from triplicate samples, (n = 3; *P < .05, ***P < .01). The pSODLUC reporter construct contains the FoxO-binding sites of the SOD promoter, whereas the pSODLUC-mut has point mutations in the first and second FoxO-binding sites and was used as a control.17 pSODLUC-mut therefore shows the basal aspecific luciferase activity. (D) Immunoblots and quantification of the nuclear fraction of β-catenin and nuclear protein (NP)–95 (loading control) from N, VE, and N+VE EC. Columns are means ± SEM, (n = 3; *P < .05, ***P < .01). (E) β-catenin luciferase activity measured by TOP/FOP assay. Results represent the mean ± SE of relative light units determined from triplicate samples, (n = 3; **P < .02). For data presented in panels A, B, and D, cells have been cultured in starving medium (see supplemental Methods) for 18-20 hours before performing the experiments.
