Fig. 2

The dual PI3K/mTOR inhibitor BEZ235 causes cell arrest at G0/G1 phase by decreasing cyclin D. a BEZ235 inhibits both PI3K and mTOR signaling in NSCLC cell lines expressing either wild-type EGFR or activating mutants of EGFR. NSCLC cells were treated with 100 nM BEZ235 for 6 h, and the levels of phosphorylated AKT, p70S6K, 4EBP1, ERK1/2, and STAT3 were examined. β-actin was used as an internal control. b BEZ235 causes cell arrest at G0/G1 phase. Cells were treated with 333 nM BEZ235 for 24 h, and then the cell-cycle distribution was analyzed by flow cytometry. The percentage of cells in the G0/G1 phase of the cell cycle was determined. Values are the results of three independent experiments and are reported as means ± SD (*, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t-test). c BEZ235 reduces expression of cyclin D1 and cyclin D3. Cells were treated as described in a. d Dual blockade of PI3K/mTOR pathways strongly diminishes cyclin D1 and cyclin D3 levels. Cells were treated for 6 h with 20 μM LY294002, rapamycin, or both, and molecular signaling was compared with that in cells treated with 100 nM BEZ235. Cell lysates were analyzed by Western blotting using the indicated antibodies. The related expression levels of protein, quantified by ImageJ as described in Materials and Methods, were shown below their corresponding blots