Hct 15 cell line

The human colorectal cancer HCT15 cell line, which demonstrates global hypermethylation, was obtained from the American Type Culture Collection . The cells were cultured at 37°C in a humidified 5% CO₂ atmosphere in RPMI 1640 medium (Invitrogen) supplemented with 10% foetal bovine serum (GIBCO). Cells in the exponential growth phase were used for subsequent experiments. For demethylation studies, cultured cells were incubated for 72 h in 0 or 50μmol/l of 5‐azacytidine (5‐Aza; Sigma‐Aldrich), a methylation inhibitor, and the medium was changed daily.

Fetal bovine serum, trypsin, and Hank’s balanced salt solution were purchased from Invitrogen (Carlsbad, CA, USA). Cell culture plates with ultralow attachment surfaces were purchased from Corning Life Sciences (Tewksbury, MA, USA). Neurobasal medium, FBS, B-27 Supplement, penicillin, and streptomycin were purchased from Life Technologies (Grand Island, NY, USA). TrypLE cell detachment solution was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The HCT-15 cell line was obtained from the American Type Culture Collection. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were obtained from EMD Millipore (Billerica, MA, USA). M2 anti-FLAG antibodies were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Antibodies against P53 (SC-6243) and OCT3/4 (SC-8628) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Antibodies against cyclin D1 (ab134175), NANOG (ab109250), SOX2 (ab59776), and TRRAP (ab73546) were purchased from Abcam (Boston, MA, USA). Antibodies against CD44 (5640), MYC-Tag (2287S), and HA-Tag (3724S) were purchased from Cell Signaling Technology (Danvers, MA, USA).