Cell Growth Assay and Inhibitor Dose-Response

For growth assays, 2 × 10³ cells were seeded on cell culture-coated 96-well plates (CellTreat). Cells were lysed with CellTitre-Glo 2.0 Reagent (Promega), and luminescence was read using a GloMax Discover Plate Reader (Promega). Cell number was assessed 2 hours after plating to account for any discrepancies in plating, and then every 24 hours for 5 days. Data were analyzed as an increase in luminescence over Day 0. For inhibitor studies, 4 × 10³ cells were seeded on cell-culture treated 96-well plates (CellTreat) to assess anchorage-dependent growth, and on ultra-low attachment 96-well plates (Corning Costar #3474) to assess anchorage-independent growth (49 (link)). On Day 4, cells were assessed using CellTitre-Glo 2.0 Reagent as described. Inhibitor data are plotted as a scaled dose-response curve for each cell line (E_drug – E_max)/(E₀ – E_max), where E_drug is the effect of the drug at a given concentration, E_max is the maximal effect of the drug, and E₀ is the effect seen in the DMSO control (72 (link), 73 (link)). Data were analyzed and IC₅₀ and AUC values were calculated using Prism 7.