Cellular proliferation assays

Proliferation of K1 and BCPAP cells was evaluated using the MTT assay (Sigma-Aldrich; Merck KGaA). Cells were plated in 96-well microtiter plates at a density of 3×10³ cells/well in a volume of 180 µl DMEM with FBS. PLX4032 with or without gefitinib was diluted in media containing 1% DMSO at 10X the final assay concentrations (PLX4032: 0.01, 0.03, 0.1, 0.3, 1 and 3 µM; gefitinib: 0.125 µM). After 24 h of culture, 20 µl each drug dilution was added in triplicate to separate wells. Cells were assayed for proliferation at 24, 48, 72, 96 and 120-h time points. The percentage of inhibition was calculated using the following formula: 100-(mean absorbance of experimental wells/mean absorbance of control wells) ×100. The half maximal inhibitory concentration (IC₅₀) values were determined by calculating the regression of plots produced from the logarithms of concentration vs. percentage inhibition, using XLfit software (version 4.2; ID Business Solutions Ltd., Guildford, UK).