Figure 6. HP1- is required for irreversible silencing of proliferation-associated genes in ACMs. (A) HP1-, -, and - are expressed in ACMs. (B) Semi- quantitative RT-PCR shows that expression of cdc2 and PLK1 are increased when HP1- is depleted in ACMs with siRNA. si NS, NS siRNA. (C) Quantitation of data in B by real-time PCR. *, P = 0.002; **, P = 0.002; ***, P = 0.007. (D) IP assay demonstrates that Rb associates with HP1- in adult mice hearts. IB, immunoblotted. (E–H) NRVMs were transfected with NS or HP1- siRNA. After 48 h in serum-free media, cells were either collected for Western blot analysis (E) or stimulated with 20% FBS for 24 h in the presence of BrdU. (F) Cells were stained for BrdU and troponin C (TnC), and nuclei were counter- stained with DAPI. Bar, 100 µm. (G and H) The percentage of BrdU-positive nuclei (G; *, P = 0.002) as well as total cell number (H; *, P = 0.038) was increased in HP1- siRNA–transfected NRVMs. Error bars represent the SEM from three replicates. deletion did not affect H3K9me3 methylation. Recent studies have suggested that, in contrast to earlier studies (Nielsen et al., 2001), Suv39h1-dependent methylation is Rb independent (Gonzalo et al., 2005; Siddiqui et al., 2007). To clarify the role of Suv39h1 on H3K9me3 levels in ACMs, siRNA was used to deplete Suv39h1 in control or IDKO cardiac myocytes. Tran- sient transfection of Suv39h1 siRNA oligonucleotides into ACMs efficiently reduced expression of Suv39h1 (Fig. 8, A and B), which 416 JCB • VOLUME 194 • NUMBER 3 • 2011