Cell viability assay and IC50 estimation

Cell viability was determined using the Cell Titer Glo assay (Promega). Cells were collected from suspension medium, spun down at 300 g for 5 min and resuspended in fresh RPMI medium and counted using a Countess automated cell counter and trypan blue (Invitrogen). 1,500 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicates on the same plate. Cells were treated with seven different concentrations of inhibitors in serial threefold-diluted TKIs or vehicle alone for a final volume of 40 μl per well. After 72 h, 11 μl of Cell Titer Glo was added to each well. Plates were shaken for 10 min, and bioluminescence was determined using a FLUOstar OPTIMA multimode microplate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO-treated cells, and normalized values were plotted in GraphPad Prism using nonlinear regression fit to normalized data with a variable slope. IC₅₀ values were calculated using GraphPad Prism at 50% inhibition. Each experiment was replicated three separate times to give biological replicates unless indicated otherwise.