Fig 5. Additive effect of KDM4D overexpression and Sav1 knockdown on ACM cell cycle activity in vivo.

(A, B, C, and D) Representative photomicrographs from iKDM4D+Sav1-sh hearts showing EdU, phospho-H3, and Aurora B immunostaing after KDM4D induction and Sav1 knockdown for 2 weeks. Nuclei were stained with DAPI (blue), cell borders with WGA (green), and cycling cells with EdU (magenta), white arrow points to the EdU⁺ CMs in (A). Nuclei were stained with DAPI (blue), CMs with cTnT (red), and cycling CM with phospho-H3 (green), white arrow points to the phospho-H3⁺ CMs in (B). (C) Aurora B (magenta, arrow) in a dividing CM. (D) Aurora B (white, arrow) in the nucleus of CMs. Nuclei were stained with DAPI (blue), CM actin with α-actinin (red), and cycling CM with Aurora B (magenta or white) in (C) and (D). Bar = 10μM. (E) Quantification of EdU⁺ ACMs in different groups. EdU⁺ CM number per mm² is shown. (F) Quantification of phospho-H3⁺ ACMs in different group. Phospho-H3⁺ CM number per mm² is shown. (G) Quantification of Aurora B⁺ ACMs in different group. Aurora B⁺ CM number per mm² is shown. Statistics: n = 3 for each group. * showed statistical significance at p<0.05 vs control in WT group; # represented statistical significance at p<0.05 vs control in iKDM4D group. One-way ANOVA followed by Tukeys’ Test.