Mccoy s 5a medium

The human colon cancer cell lines, HCT116 and HT-29, were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and were maintained in McCoy's 5A medium (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (100 U/mL and 100 μg/mL; Invitrogen, Carlsbad, CA, USA). The cells were cultured in humidified air at 37°C with 5% CO₂. BC was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

The human colon cancer cell line, HCT116 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HCT116 cells were double-stained with CD133 and CD44 monoclonal antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) and sorted with a FACSAria flow cytometer (BD, Franklin Lakes, NJ, USA). CD133⁺CD44⁺ HCT116 CSCs were maintained in McCoy’s 5A Medium (Welgene, Daegu, Korea) supplemented with 10% FBS (Gibco, Gaithersburg, MD, USA) and 1% penicillin streptomycin (100 U/mL and 100 μg/mL, respectively; Invitrogen, Carlsbad, CA, USA). BC (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in tetrahydrofuran (Sigma-Aldrich) under dim light. Concentration of 20 μM BC (BC 20) and 40 μM BC (BC 40) were applied to HCT116 cells for 6 days. Following the dissolution of 5-aza-2′-deoxycytidine (AZA; Sigma-Aldrich) in dimethylsulphoxide (DMSO; Sigma Aldrich), this solution was incubated with cells for 3 days. Cultured medium was replaced every 48 hours with fresh media and BC.

Human colorectal carcinoma HCT116, SW480, and LoVo cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). HCT116, SW480 cells were maintained in McCoy’s 5A Medium (Welgene, Gyeongsan, Korea) and LoVo cell was maintained in Roswell Park Memorial Institute Medium (RPMI 1640, Welgene) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 1% penicillin-streptomycin (Gibco) and 1% Sodium Pyruvate (Gibco) at 37°C in an incubator containing 5% CO₂ atmosphere.