Phenotypic analysis and in vitro sensitivity of SKBR3, JIMT-1 and MCF7-HER2 cells to gefitinib, RAD001, or the gefitinib and RAD001 combination. (A) Expression of HER2, EGFR, HER3, P-AKT and AKT analyzed by Western blotting. (B) Cells were treated for 72 or 144 h with gefitinib (Gef), RAD001 (RAD) and the gefitinib and RAD001 combination at 200:1 molar ratio (Gef:RAD). Cell viability curves were plotted based on data obtained with an Alamar Blue assay and reported as Fa (fraction of cells affected by the treatment, where 1 is equivalent to 100% cytotoxicity). Each data point represents the mean ± SD of 3 wells. Asterisks above data points indicate significantly (p < 0.05) greater Fa in the combination treated cells compared to the single drugs. Achievable levels of gefitinib and rapamycin analogs reported in human blood are ~ 1 μM and 5-15 nM, respectively. All cell lines were screened 3 times with single drugs and 2-3 times with the drug combinations to assure consistency. The results presented are from a representative experiment. (C) The combination index (CI) was calculated with the CompuSyn™ software based on data derived from Alamar Blue assay completed in cells treated with the single drugs and drug combinations at the indicated molar ratios and plotted versus Fa. Each data point moving from left to right for any given ratio of gefitinib to RAD001 represents an effect of increasing drug concentrations. The data points below CI values of 0.8, denoted by a horizontal line on each plot, are indicative of synergistic interactions.