iMEF cells were grown in DMEM supplemented with 10% FCS, 100 mM nonessential amino acids, 1 mM L-glutamine. Ezh2flox/flox;ROSA26-CreERT2 or Ezh2flox/Δ;ROSA26-CreERT2 MEF cells were isolated from 13.5-d-old embryos and subsequently infected with the following retroviral constructs: pMXs-hc-MYC (Addgene, 17220) to generate c-Myc iMEFs, pBABE-hygro p53 DD (Addgene, 9058) to generate p53-DN iMEFs, and Ndy1-MigR1 (kindly provided by Philip N. Tsichlis) to generate Ndy1 iMEFs. A clone was obtained by limiting dilution of a pool of c-Myc Ezh2flox/Δ;ROSA26-CreERT2 iMEFs. For conditional deletion of Ezh2, cells were treated with 4-hydroxytamoxifen (Sigma) at a final concentration ranging from 1 nM to 1 µM. For Ezh1 and Ezh2 rescue experiments, cells were infected with MSCVhygro-Flag-Ezh1 or MSCVhygro-Flag-Ezh2 ecotropic retroviruses.
The Myc-CaP mouse prostate cancer cell line was generously provided by Charles L. Sawyers, and cells were grown in DMEM supplemented with 10% FCS, 100 mM nonessential amino acids, and 1 mM L-glutamine. To obtain optimal growth conditions, the growth medium was supplemented with a growth factor cocktail composed of 25 µg/mL bovine pituitary extract (Life Technologies), 5 µg/mL bovine insulin (Sigma), and 6 ng/mL recombinant human epidermal growth factor (Sigma).
The Myc-CaP mouse prostate cancer cell line was generously provided by Charles L. Sawyers, and cells were grown in DMEM supplemented with 10% FCS, 100 mM nonessential amino acids, and 1 mM L-glutamine. To obtain optimal growth conditions, the growth medium was supplemented with a growth factor cocktail composed of 25 µg/mL bovine pituitary extract (Life Technologies), 5 µg/mL bovine insulin (Sigma), and 6 ng/mL recombinant human epidermal growth factor (Sigma).