Rb and p130 play an overlapping role in the silencing of proliferation-promoting genes and the maintaining of heterochromatin in ACMs. (A) Reexpression of G2/M and cytokinesis genes in IDKO hearts. Semiquantitative RT-PCR was performed on total RNA isolated from ventricular tissue from mice with the indicated genotypes. For quantitation of changes in gene expression, see Fig. S4 A. AurKB, Aurora kinase B. (B) Western blotting demonstrates that H3K9me3 and H3K27me3 levels are unchanged in IDKO ventricles. Also see Fig. S4 (B and C) for H3K9me3 and H3K27me3 levels in isolated ACMs determined by immunostaining. (C) Heterochromatin was disrupted only in ACMs from IDKO mice. Confocal microscopy was performed on myocardial sections (red, troponin C [TnC]; blue, DAPI). Bar, 10 µm. (D) Percentages of heterochromatin-positive nuclei were quantified in ACMs isolated from hearts with the indicated genotypes and treatment (n = 3 per group; *, P < 0.0001). Error bars represent the SEM. CM, cardiac myocyte. (E and F) H3K9me3 (E) and H3K27me3 (F) levels at E2F-dependent promoters remained unchanged in IDKO ACMs, as analyzed by ChIP using chromatin extracts from purified myocytes.