Assessment of cell death, apoptosis and cell cycle in SKBR3, JIMT-1 and MCF7-HER2 cells treated with gefitinib, RAD001 and the gefitinib and RAD001 combination used at 200:1 (Gef:RAD) molar ratio. (A) Assessment of cell death by HCS. Cells were seeded in 96-well plates and treated with indicated drug concentration. After 72 h cells were stained in situ with DRAQ5 (stain for viable cells) and ethidium homodimer (stain for dead cells) and images were acquired with IN Cell 1000. The imaging data were analyzed with the IN Cell 1000 Investigator software and the results are expressed as percentage of dead cells relative to DMSO control. Asterisks above data points indicate a significantly (p < 0.05) greater percentage of dead cells in the combination treated cells compared to cells treated with gefitinib or RAD001 alone. Achievable levels of gefitinib and rapamycin analogs reported in human blood are ~ 1 μM and 5-15 nM, respectively. Each data point represents the mean ± SD from 3 replicate wells. Data from representative experiments are shown. (B - C) Flow cytometric analysis of apoptosis (B) and cell cycle (C) in cells treated with 1 μM gefitinib (Gef), 5 nM RAD001 (RAD) or the combination of both drugs at 200:1 (Gef:RAD) molar ratio. Each bar represents a mean ± SD from 3 replicate samples. Asterisks indicate a significant difference (p < 0.05) between cells treated with the gefitinib and RAD001 combination compared to the single drugs. Representative experiments are shown.