HP1-γ is required for irreversible silencing of proliferation-associated genes in ACMs. (A) HP1-α, -β, and -γ are expressed in ACMs. (B) Semiquantitative RT-PCR shows that expression of cdc2 and PLK1 are increased when HP1-γ 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-γ in adult mice hearts. IB, immunoblotted. (E–H) NRVMs were transfected with NS or HP1-γ 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 counterstained 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-γ siRNA–transfected NRVMs. Error bars represent the SEM from three replicates.