Combination Therapy Impacts TNBC Cell Viability

The effects of vehicle (DMSO), EC359, and HDACi alone or in combination on cell viability were measured using the MTT cell viability assay^{16 (link)}. HDACi and EC359 concentrations used in the assays were based on our earlier studies^{16 (link)}, dose–response curves using TNBC models, and published studies^{15 (link)}. The combination index (CI) of EC359 + HDACi therapy was determined using Chou-Talalay method^{37 (link)}. Apoptosis was measured using Annexin V-PI staining (BioLegend, San Diego, CA) and Caspase-Glo® 3/7 assays (Promega, Madison, WI) according to manufacturer’s protocol. For colony formation assays, TNBC model cells (500 cells/well) were seeded in triplicate in 6-well plates, treated with indicated drugs for 5 days, and allowed to grow for 14 days. The cells were fixed in ice-cold methanol and stained with 0.5% crystal violet solution. Colonies that contain ≥50 cells were counted and used in the analysis. The effect of combination therapy on cell invasion was studied using the Corning® BioCoat™ Growth Factor Reduced Matrigel® Invasion Chamber assay (Corning, Corning, NY). MDA-MB-231 and BT-549 cells were treated with vehicle or EC359 or HDACi or combination for 22 h and invaded cells were determined and quantitated using the manufacturer^,s protocol.