EGFR mutant cells downregulate Mcl-1 in response to PI3K-mTOR inhibition. (A) EGFR mutant cells (HCC827, H1975, and HCC827 GR6) and HER2-amplified cells (SKBR3 and BT474) were treated with either DMSO control or the indicated drug(s) (TKIs 1 μM, NVP-BEZ235 0.2 μM, AZD6244 0.2 μM for SKBR3 cells, 1 μM for HCC827 and HCC827 GR6 cells, and 2 μM for H1975 and BT474 cells) and 72 h later cells were subjected to flow cytometry to determine subG0/G1 population and the distribution of cycling cells, as described in Materials and Methods. Data are presented as mean ± S.D. of triplicate experiments. (TKI is CL-387,785 (CL) for H1975 cells and gefitinib/PHA-665752 (gef/PHA) for HCC827 GR6 cells). Statistical analyses comparing BEZ versus BEZ/AZD were P < 0.001 for HCC827, H1975, HCC827 GR6, and were not significant (P > 0.01) for BT474 an SKBR3. (B) Cells were treated with either DMSO (-) or drug for 30 h. Cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. The SKBR3 cells were treated for 72 h with the indicated drugs because no substantial PARP cleavage was observed after 30 h with any of the drug treatments.