Fig. 2

Neutralizing N-cadherin signaling results in translocation of β-catenin to the cell nuclei. Matured mouse cardiomyocytes were isolated via FACS using VCAM-1 antibody. a Representative images of untreated control and N-cadherin antibody (1:1000)-treated matured mouse cardiomyocytes immunostained for membrane-bound β-catenin (red). Cell nuclei (blue) were stained with DAPI. Scale bar, 50 μm. b Graphical representation of the percentage of cells with high membrane-bound β-catenin. A cut-off value > 150 was imposed to remove background. N-cadherin antibody-treated cardiomyocytes showed reduced amount of membrane-bound β-catenin (~ 50%) compared to the untreated control. Error bars indicate s.d., n = 3 experiments, **P < 0.01, evaluated by Student’s t test. c Representative images of untreated control and N-cadherin antibody (1:1000)-treated matured mouse cardiomyocytes immunostained for nuclear β-catenin (green). Cell nuclei (blue) were stained with DAPI. Scale bar, 50 μm. d Graphical representation of the percentage of cells with high nuclear β-catenin. A cut-off value > 300 was imposed to remove background. N-cadherin antibody-treated cardiomyocytes showed a 1-fold increase in the amount of nuclear β-catenin compared to the untreated control. Error bars indicate s.d., n = 3 experiments, **P < 0.01, evaluated by Student’s t test. e Quantitative PCR analysis of mRNA isolated from cardiomyocytes from untreated control and cardiomyocytes treated with N-cadherin antibody (1:1000). The relative gene expression levels showed that most genes in canonical Wnt signaling pathway were significantly upregulated in cardiomyocytes treated with N-cadherin antibody. Error bars indicate s.d., n = 3 experiments. *P < 0.05 and **P < 0.01, evaluated by Student’s t test. f Quantitative PCR analysis of mRNA isolated from cardiomyocytes in untreated control and cardiomyocytes treated with N-cadherin antibody (1:1000) or CHIR99021 (1 μM). The relative expression levels of Wnt signaling effector genes (Axin2 and Lef1) were upregulated in cardiomyocytes treated with either N-cadherin antibody or CHIR99021. Error bars indicate s.d., n = 3 experiments. **P < 0.01 for Kruskal-Wallis one-way analysis of variance compared to control