Immunofluorescence staining

Immunofluorescence staining was conducted as described previously [56]. The cells in four-well chamber slides were fixed for 10 min at room temperature (RT) with 4% paraformaldehyde, and subsequently with cold methanol at −20 °C for 5 min. The slides were then treated with 0.2% Triton X-100 for 5 min at RT and blocked with 2% bovine serum albumin overnight at 4 °C. For H3K9me2 and cyclin D1, G9a and cyclin D1 co-staining in MEFs the primary antibodies used were mouse monoclonal anti-H3K9me2 (ab1220) (Abcam Inc.) (1/800), mouse monoclonal anti-G9a (clonal A8620A) (PP-A8620A-00) (R&D System) (1/500), and rabbit polyclonal anti-cyclin D1 (clone H-295) (sc-753) (Santa Cruz Biotechnology, Santa Cruz, CA) (1/200). The secondary antibodies used were Alexa Fluor 568-conjugated goat anti-mouse immunoglobulin G (IgG) (Molecular Probes, Inc.) (1/500) and Alexa Fluor 647-conjugated F(ab′)2 fragment of goat anti-rabbit IgG (Molecular Probes, Inc.) (1/500). For HT1080 immunostaining the primary antibodies used were mouse monoclonal anti-cyclin D1 (clone DCS-6) (sc-20044) (Santa Cruz Biotechnology, Santa Cruz, CA) (1/200), rabbit polyclonal anti-Lamin B1 (ab16048) (Abcam Inc.) (1/900), and mouse monoclonal anti-H3K9me2 (ab1220) (Abcam Inc.) (1/800). The secondary antibodies used were rhodamine-conjugated F(ab′)2 fragment of goat anti-rabbit IgG (Jackson Immuno Research Laboratories, Inc.) (1/500) and Alexa Fluor 633-conjugated F(ab′)2 fragment of goat anti-mouse IgG (Molecular Probes, Inc.) (1/500). The samples were visualized on a Zeiss LSM 510 META Confocal Microscope with a ×63 objective.