Blockade of phosphoinositide 3-kinase, but not additional HER2 blockade, inhibits phosphoinositide 3-kinase signaling in lapatinib-resistant cells with PIK3CA mutation. (A) Lapatinib-resistant cells in the presence of lapatinib were treated with trastuzumab overnight, with BKM120 or with the combinations as indicated, and lysates were analyzed by immunoblotting. (B) LR cell lines were plated in 96-well plates. Twenty-four hours after plating, cells were treated with inhibitors as indicated and fixed after seventy-two hours of treatment (lap = lapatinib, tras = trastuzumab, pert = pertuzumab and BKM = BKM120). Cells were stained with sulforhodamine B (SRB). Mean SRB absorbance normalized to cells treated with lapatinib only is shown (duplicates from two separate experiments; bars = SEM). (C) Cells were treated as described in (B), and apoptosis was measured by luminescence after 24-hour treatment with the Caspase-Glo reagent. Mean luminescence (duplicates from two experiments) normalized to lapatinib only is displayed (bars = SEM).