M3 media

Human colorectal cancer cell lines, including HCT116, HT29, RKO, DLD-1, SW403, SW1463, DiFi, and NCI-H508, were purchased from the ATCC and cultured in McCoy's 5A modified media. SNU-407 was purchased from AddexBio and cultured in RPMI1640 (Invitrogen). NCM356D non-transformed colonic epithelial cell line was purchased from INCELL and cultured in M3 media (INCELL). DR5-knockout (KO), PUMA-KO, and p53-KO HCT116 cells were described previously (20 (link), 27 (link)). Cells were authenticated by genotyping and analysis of protein expression by Western blotting, and routinely checked for Mycoplasma contamination by PCR. All cell lines were maintained at 37°C and 5% CO₂ atmosphere. Cell culture media were supplemented with 10% defined FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. For drug treatment, cells were plated in 12-well plates at 20%–30% density 24 hours before treatment. DMSO stocks of NEO2734, CPI-637, I-BET151, OTX015, 5-FU, oxaliplatin, and SN-38 were prepared and diluted in cell culture media before adding to cells. All drugs were purchased from commercial sources, except for NEO2734 and CPI-637 which were provided by NEOMED Therapeutics 1, Inc.

NCM460 cells were purchased from INCELL Corporation (San Antonio, TX, USA) and cultured in M3 media (San Antonio, TX, USA) supplemented with 10% FBS (fetal bovine serum), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a 95% humidified atmosphere with 5% CO₂. Cells were passaged at pre-confluent densities using 0.05% trypsin and 0.5 mm EDTA (Invitrogen, Karlsbad, Germany).