b Actanti caspase 2 at 1:1000. A monoclonal mouse b Actin was utilized as a control to standardize sample loading. Detection of proteins was Evodiamine Isoevodiamine done with peroxidase conjugated anti mouse or anti rabbit IgG secondary antibodies and ECL. Antineoplastic Agents and Cell Viability Assays For cell viability assays, 16103 cells were plated in triplicate in a 96 well microtiter plate and allowed to grow for 24 hours to an approximate confluence of 30 . For drug inhibition studies, NVP BEZ235 and NVP BKM120 and LY294002 were used to treat lung cells at various concentrations. For combination experiments, the mTORC1 inhibitor, rapamycin and EGFR inhibitor erlotinib were utilized.
Cell viability was evaluated at 72 hours using the CellTiter GloTM Luminescent GW 791343 Cell Viability Assay, according to the manufacturer,s instructions, an equal volume of the Cell Titer GloTM reagent was added to the wells and after a 10 minute incubation, luminescence was recorded using a VictorTMX multilabel plate reader. The IC50 values were determined by the XLfit software. Synergistic Drug Effect Analysis The Chou and Talalay combination index analysis method was utilized to analyze results from combinatory drug experiments. NVP BKM120 and LY294002 were used in combination with the mTOR inhibitor, rapamycin. NVP BEZ235 was combined with erlotinib, at their respective single drug IC50 or IC25 concentrations. The IC50 and IC 25 values of each drug were first obtained from single drug viability assays and then utilized to design drug combination experiments.
The level of synergism was assessed over a wide range of drug concentrations, focusing on concentrations below or at the IC50 for either drug alone. Our results were analyzed for synergistic, additive, or antagonistic effects using CalcuSyn software. Synergistic effect is indicated by a Combination Index of less than 0.9, additive effect by a CI between 0.9 and 1.1, and antagonistic effect by CI greater than 1.1. Results Expression of PI3K p85 and p110a? subunits in human lung cancer specimens For the MTMA, 166 and 190 specimens were fully assessable with regard to PI3K p85 and p110a subunit expression, respectively. PI3K did not show significant nuclear staining for either subunit, and we therefore analyzed only the cytoplasmic compartment. Staining patterns within the tumor mask within a histospot were highly homogenous for both subunits.
AQUA scores ranged from 7.12 to 127.04 for the p85 subunit, and from 5.43 to 138.56 for the p110a subunit. An example of AQUA staining of histospots for p85 and p110a expression is shown in Figure 1 and Table S2. Histospots were deemed uninterpretable if they had insufficient tumor cells, loss of tissue in the spot, or an abundance of necrotic tissue. Specimens with less than 5 tumor area per spot were not included in the AQUA analysis. We assessed the associations between PI3K subunit expression and histologic subtype, by unpaired t tests. Expression of both the p85 and the p110a subunits were signif