CNOT4 regulates KDM5 stability by inducing its polyubiquitin-mediated degradation in cardioyocytes. (A) Analysis of in situ PLAs in cardiomyocytes. KDM5 and CNOT4 interactions (indicated by dots) were visualized, and nuclei were counterstained with DAPI. Scale bar: 20 μm. (B) Co-IP of KDM5 with CNOT4 in cardiac lysates from adult mice. IgG served as a negative control. (C) Representative western blot images and statistical analysis of KDM5 and CNOT4 protein expressions in cardiomyocyte extracts in the NC-siRNA and CNOT4-siRNA groups (n = 3 per group). (D) Representative western blot images of polyubiquitinated KDM5 in the NC-siRNA and CNOT4-siRNA groups, with KDM5 polyubiquitination levels being significantly decreased in CNOT4-siRNA–transfected cardiomyocytes compared with the NC-siRNA cells. (E) Representative western blot images of the polyubiquitinated KDM5-flag in HEK293 cells from the indicated groups. (F) Representative western blot image and its quantification of KDM5 protein expression in HEK293 cells in the indicated groups (n = 3 per group). (G) Co-IP of CNOT4-HA with KDM5-WT-flag or KDM5-Mut-flag in lysates from HEK293 cells. Data represent mean ± SEM. Student’s or Welch’s t-tests were used for comparisons between two groups when the variances were equal or unequal, respectively. ^###P < 0.001, with comparisons indicated by lines.