Persistent activation of PI3K signaling promotes survival in lapatinib-resistant cells. (A) pHER2, total HER2, AktT308, AktS473, p70S6KS371, 4EBP1S65, and survivin steady-state protein expression in untreated parental BT474, BT474 treated with 0.5 μM lapatinib for 48 hours, and rBT474 maintained in 1 μM lapatinib, as determined by Western blot analysis from whole cell extracts. (B) Phospho-PI3K protein expression was determined by RPMA in the same treatment groups as described in (A). Results represent the mean ± standard error of triplicate samples, and are representative of three independent experiments. *P < 0.0018. (C) Molecular knockdown of PI3K by using pooled siRNA against PI3K subunits (*) in rBT474 cells was confirmed by Western blot analysis by using subunit-specific antibodies and an anti-PARP cleavage-product antibody. Cells transfected with scrambled siRNA construct (NSC) served as controls. Actin steady-state protein levels served as a control to ensure equal loading of protein. The results are representative of three independent experiments. (D) The effects of siRNA-mediated knockdown of PI3K on rBT474 cell growth (P < 0.0058). Nonspecific siRNA construct (NSC) served as a control. Results represent the mean ± standard error of triplicate samples, and are representative of three independent experiments.