Fig. 6: Targets screening for FoxO3-mediated cardiac regeneration in postnatal mice.

a RNA-seq identified 4 overlapping genes (Trp53, Sfrp2, Cdkn2c, and Cdkn2d) between the downregulated gene sets for negative regulation of cell proliferation (NRP) and growth (NRG) in CKO ventricles versus controls at p1 and p14. b ChIP-qPCR validation of FoxO3 binding to the promoters of the identified 4 potential targets in primary cardiomyocytes isolated from sham and injured hearts at 5 dpr (n = 3). c Fold enrichment of FoxO3 signal relative to IgG signal for these 4 potential target promoters in cardiomyocytes isolated from sham-operated hearts (n = 3). d The qPCR validation of these 4 potential targets expression in control and CKO cardiomyocytes at 5 dpr (n = 3). e, f GSEA analysis based on the RNA-seq data revealed that Trp53 and Sfrp2 pathways are associated with FoxO3-mediated postnatal heart regeneration. g The interactions of FoxO3 with the promoters of Trp53 and Sfrp2 are evaluated using reporter gene and mutation assay (n = 5). h–j Primary cardiomyocytes isolated from CKO neonatal mice at p1 are transfected with Adv5-NC and Adv5-Sfrp2 adenoviruses for 48 h, followed by immunofluorescent staining for cTnT to verify cardiomyocytes, for proliferation markers Ki67 (h), EdU (i), and pH3 (j) to verify proliferating activity of cardiomyocytes. Representative images (left) and quantification (right) of proliferating cardiomyocytes in Adv5-NC and Adv5-Sfrp2 groups are shown (n = 8). All data are presented as the mean ± SEM. P values are from two-tailed t test (h–j), one-way ANOVA followed by Tukey’s multiple comparisons test (b, c, g) or two-way ANOVA followed by Sidak’s multiple comparisons test (d). ns, no significant difference. Source data are provided as a Source Data file.