Fig. 4: The pro-mitotic effects mediated by N-Cadherin in CMs depend on its interaction with β-Catenin.

a The protein half-life of β-catenin upon Cdh2/N-Cadherin knockdown in mouse HL-1 CMs was determined using a cycloheximide chase assay, which revealed an accelerated degradation of β-Catenin in Cdh2 KD, compared to Scramble control, CMs (n = 7 in each group at various time points). A two-way ANOVA test (Interaction p = 0.0001; Time points p < 0.0001; and Group p < 0.0001) was utilized followed by a Bonferroni’s multiple comparison test. b Co-immunoprecipitation (Co-IP) experiments using neonatal mouse cardiac lysates showed that N-Cadherin could be pulled down with β-Catenin, indicating their physical interaction (left panel). The Co-IP experiments were performed in three independent biological replicates. Schematic illustration of the functional domains of N-Cadherin protein. The β-Catenin binding motif (BBM) locates at the C-terminus of N-Cadherin. Cdh2: wild-type N-Cadherin; ΔCdh2: c-terminus-truncated N-Cadherin. c Immunoblotting revealed that β-Catenin was markedly increased in Cdh2-, but not ΔCdh2-, overexpressed mouse neonatal CMs (n = 5 for Ctrl; n = 5 for Cdh2 OE; and n = 4 for ΔCdh2 OE mouse neonatal CMs). Quantification of N-Cadherin and β-Catenin proteins was shown on the right. A one-way ANOVA test (p = 0.0306 in relative N-Cadherin expression; and p < 0.0001 in relative β-Catenin expression) was utilized followed by a Fisher’s LSD post-test correction. d Overexpression of WT, but not C-terminus-truncated (ΔCdh2), N-Cadherin led to increased neonatal CMs proliferation, suggesting that BBM is required for the promo-mitotic effects of N-Cadherin in CMs (n = 4 for Ctrl; n = 3 for Cdh2 OE; and n = 4 for ΔCdh2 OE mouse neonatal CMs). A one-way ANOVA test (p = 0.0059) was utilized followed by a Tukey post-test correction. All experimental data are presented as means ± SEM. Exact p values are indicated in figures.