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. Author manuscript; available in PMC: 2014 Jan 18.
Published in final edited form as: Circ Res. 2012 Nov 20;112(2):257–266. doi: 10.1161/CIRCRESAHA.112.275818

Figure 6. Treatment of differentiating mESCs with Wnt3a results in reduced cardiomyogenesis associated with increased exclusion of endogenous Nkx2.5 from the nucleus.

Figure 6

Differentiating D3 mESCs were treated with Wnt3a or vehicle (PBS + 0.1% BSA) control from days 4 to 7. A) Wnt3a- and vehicle- treated cells were fixed on day 7 and labelled with antibodies specific to Mlc2v and stained with Hoechst dye to detect nuclei. Scale bar represents 10μm. B) The number of Mlc2v+ cells was quantified in vehicle- and Wnt3a-treated cultures (n=3, *p<0.05). C) The expression of genes associated with cardiomyogenesis was analyzed by QPCR in vehicle- and Wnt3a-treated cultures. Data are expressed relative to expression levels in vehicle treated cultures (n=3, *p<0.05). D) Total protein was harvested from Wnt3a- and vehicle- treated mESCs on day 7 and analyzed by western blot with antibodies specific to the factors indicated. E) The exclusion of endogenous Nkx2.5 from the nucleus was quantified in vehicle- and Wnt3a-treated mESC cultures (n=3, *p<0.05). F) Differentiated mESCs from vehicle- and Wnt3a-treated cultures were fixed on day 7 and labelled with antibodies specific to Nkx2.5 and Mlc2v. Scale bar represents 10μm. G) Differentiated mESCs from Wnt3a-treated cultures were fixed on day 7 and labelled with antibodies specific to Nkx2.5 and pMypt1. Scale bar represents 10μm.