Figure 3.

Impaired differentiation of VE-cadherin-KO ESCs towards cardiac and endothelial cells

(A) VE-CADHERIN AJs in KO and E14T clones on day 10 shown by immunofluorescence and qPCR on days 4–13. Note the absence of VE-CADHERIN in KO EBs.

(B) Immunofluorescence staining in KO and E14T EBs showing downregulation of ISL1 and FLK1 but not N-CADHERIN.

(C) Flow cytometry analysis (pseudocolor dot plots) for expression of PDGFRa and FLK1 in VE-CADHERIN KO EBs on days 4 and 5 compared with control EBs.

(D) isl1, flk1, n-cadherin, tbx5, fgf8, nkx2.5, ctnt, mlc2v, and mlc2a mRNA expression levels in KO and control EBs quantified by real-time qPCR during the indicated differentiation days (three independent biological experiments, n = 3). Data represent mean ± SEM. The statistical significance of difference was determined by two-way ANOVA with Dunnett’s multiple comparisons as post hoc analysis.

(E) cTNT and VE-CADHERIN expression in KO and control EBs on day 10.

(F and G) Immunostaining for the endothelial markers PECAM-1 and vWF on day 10 in KO EBs compared with the control.

Scale bars, 20 μm. All experiments were performed at least three times, and representative data are shown.