I bet762

FRO (purchased from the European Collection of Cell Cultures, Salisbury, United Kingdom) and SW1736 (obtained from Cell Lines Service GmbH, Eppelheim, Germany) are human cell lines derived from ATC, both harboring a BRAF V600E mutation (Pilli et al. 2009 (link); Schweppe et al. 2008 (link)). Both cell lines have been tested for being mycoplasma-free and authenticated by STR analysis to be appropriate cell lines of thyroid cancer origin. FRO and SW1736 were grown in DMEM medium (EuroClone, Milan, Italy) and RPMI 1640 medium (EuroClone) respectively, supplemented with 10% fetal bovine serum (Gibco Invitrogen, Milan, Italy), 2 mM L-glutamine (EuroClone) and 50 mg/ml gentamicin (Gibco Invitrogen), in a humidified incubator (5% CO₂ in air at 37°C). Cultured cells were treated with vehicle (DMSO, Sigma Aldrich, Saint Louis, MO, USA) or the following agents: JQ1 (50 nM–10 μM in DMSO) (Cayman Chemical, Ann Arbor, MI, USA) and I-BET762 (50 nM –10 μM in DMSO) (Merck Millipore, Darmstadt, Germany). T4888M and T3531L are ATC cell lines derived from tumors developed by [Pten, Tp53]^thyr−/− mice (Antico Arciuch et al. 2011 (link)). Cells were grown in DMEM supplemented with 10% FBS.

Human CRC cell lines, including HCT116, HT29, RKO, SW48, Lim1215, and NCI-H508, were purchased from the American Type Culture Collection and cultured in McCoy’s 5A modified media (Invitrogen). SNU-407 was purchased from AddexBio and cultured in RPMI 1640 (Invitrogen). DR5-knockout (KO) HCT116 cells were previously described (26 (link)). DR5-KO RKO cells were generated by CRISPR/Cas9 using a single guide RNA sequence 5’-CGCGGCGACAACGAGCACAA-3’ as described (27 (link)). Cells were authenticated in 2018 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 (HyClone), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For drug treatment, cells were plated in 12-well plates at 20–30% density 24 hr before treatment. DMSO (Sigma) stocks of JQ1, I-BET151 (Apexbio), OTX015, I-BET762, 5-FU, and oxaliplatin (Sigma) were prepared and diluted in cell culture media before adding to cells.