Gsk2801

Cells were seeded in 96 well plates (MDA-MB-231 cells, 5000 cells/well; HCC-1806 and SUM-159 cells, 8000 cells/well) in triplicate with 200 µl of media per well and allowed to adhere for 24 h [47 (link)]. JQ1 (SML1524) and GSK2801 (SML0768) were purchased from Sigma- Aldrich and dissolved in DMSO to make stock solutions (JQ1-20 mg/mL; GSK2801-10 mg/mL) and further dilutions were made with culture media immediately prior to each experiment. JQ1 and GSK2801 stock solutions were stored at 4 oC and -20 oC, respectively for no longer than 30 days. The spent medium was aspirated and the fresh medium containing drug concentrations ranging from 0.125 µM – 20 µM were used to treat the cells along with DMSO control. The combined treatments were carried out with JQ1 (25 nM, 50 nM, 125 nM, 250 nM, 500 nM, 1000 nM) combined with 10 µM and 20 µM of GSK2801 to assess the possible synergistic effects. After 72 h, spent media was aspirated; MTT was added and incubated for 3 h at 37 °C. MTT was removed, DMSO was added, and the plate was kept on shaker for 30 min at 60 RPM to dissolve crystals completely. The plate was read at 540 nm and OD recorded. Percent viability was calculated $(ODtreated÷ODControl)\times 100$ and the viabilities were plotted [48 (link)]. The combination index (CI) for each treatment combination was determined by the Chou-Talalay method [49 (link)] using CompuSyn software [50 ].

GSK2801, JQ1, UNC0642, UNC1999, BAY 598, GSK864, GSK484 and Tubastatin were provided by Sigma-Aldrich (St. Louis, MO, USA); TP-472 (Structural Genomics Consortium); 5-Azacytidine and SAHA from Abcam (Cambridge, UK); CN26, CN101, CN133, CN147, and C161 (Hooker laboratory). Unless otherwise indicated, all compounds were dissolved in DMSO.