Human CRC cell lines, including HCT116, RKO and HT29 (purchased from American Type Culture Collection) and derivatives, were cultured in McCoy’s 5A (Gibco; ThermoFisher) modified media supplemented with 10% defined FBS (Hyclone), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). NCM356 non-transformed colonic epithelial cells (INCELL) and derivatives were cultured in M3 Media (INCELL). DR5 KO HCT116 and RKO cells were described previously [49 (link)].
Cell lines 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 an atmosphere of 5% CO2.
For drug treatment, cells were plated in 12-well plates at 20-30% density 24 hr prior to treatment. Chemical agents, including sulindac sulfide (Merck), indomethacin (Sigma), celecoxib (Sigma), acetaminophen (Sigma), metformin (Cayman Chemical), diclofenac (sodium salt; Cayman Chemical), sodium salicylate (Sigma), and salubrinal (Apex Bio), were prepared as DMSO stock solutions, except for Naproxen sodium (Apex Bio), which was dissolved in dH2O. Stock solution of each agent was diluted to appropriate concentrations in cell culture medium, and then added to cells.
Cell lines 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 an atmosphere of 5% CO2.
For drug treatment, cells were plated in 12-well plates at 20-30% density 24 hr prior to treatment. Chemical agents, including sulindac sulfide (Merck), indomethacin (Sigma), celecoxib (Sigma), acetaminophen (Sigma), metformin (Cayman Chemical), diclofenac (sodium salt; Cayman Chemical), sodium salicylate (Sigma), and salubrinal (Apex Bio), were prepared as DMSO stock solutions, except for Naproxen sodium (Apex Bio), which was dissolved in dH2O. Stock solution of each agent was diluted to appropriate concentrations in cell culture medium, and then added to cells.