Inhibition of phosphatidylinositol 3-kinase/mammalian target of rapamycin abrogates lapatinib resistance in HER2+ breast cancer cells. (a) BT-474 and SKBR3 cells were serum-starved for 3 days, then treated with or without 1 μM lapatinib or 250 nM BEZ235 for 2 days. Cells were fixed, stained using the ApoBrdU kit (Phoenix Flow Systems), and analyzed by flow cytometry. Cells were considered apoptotic if they exhibited sub-G1 levels by propidium iodide staining, and/or high fluorescein isothiocyanate (FITC)-bromodeoxyuridine (BrdU) labeling. Representative plots are shown, and the percentage of apoptotic cells (mean of triplicates ± standard deviation) is noted in each panel; blue, live; red, dead. (b) BT474 and SKBR3 cells were selected for long-term growth in the presence of 2 μM lapatinib to generate resistant cells . Parental and lapatinib-resistant cells were treated with or without 250 nM BEZ235 in growth medium (lapatinib-resistant cells were maintained in 2 μM lapatinib). Media and drugs were replenished every 2 to 3 days. Cell viability was measured after 5 to 6 days by WST1 assay (Roche). Data are presented as percentage parental control for each cell line, mean of triplicates ± standard deviation. *P < 0.05 by Bonferroni post-hoc test compared to parental control.