Reproducible Drug Combination Assay

Indomethacin and GW9662 were purchased from Cayman Chemical (Ann Arbor, MI, USA), and methoctramine was purchased from SigmaAldrich (St. Louis, MO, USA). All of the drugs were dissolved in dimethyl sulfoxide (DMSO). DMSO was pumped with nitrogen for one minute before using it to avoid drug oxidation. Aliquots of drugs were stored at −80 °C until used and discarded after the thawing. For all experiments, the maximal concentration of DMSO in the culture medium was 0.5%, which did not influence cell viability. For immunoblotting, the cells were exposed to the drugs for 24 h, as previously described [19 (link)]. For the cell viability and combination studies, the cells were exposed to at least six concentrations of each drug for 72 h, as described in the results section.