The effects of the PI3K/mTOR inhibitor NVP-BEZ235 on SHEP NB cells.

<p><b>A</b> Cells were either left untreated, treated for 24 µM PI-103, a well-characterized pan-PI3K inhibitor used as positive control, or the indicated concentrations of NVP-BEZ235. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein served as surrogate read-outs for PI3K and mTOR activity, respectively, and were analyzed by Western blotting, β-actin served as loading control. <b>B</b> Cells were either left untreated, treated for 24 hrs with either 0.6 µM PI-103 as positive control, or 0.6 µM NVP-BEZ235 for the indicated lengths of time. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein were analyzed by Western blotting, β-actin served as loading control. <b>C</b> Cells were either left untreated, treated for indicated length of time with 0.6 µM NVP-BEZ235, or treated for 24 hrs with 0.2 µg/ml doxorubicin as positive control. Protein expression levels and cleavage of caspase-.3 protein were analyzed by Western blotting, β-actin served as loading control. <b>D</b> Cells were cultured either in the presence or absence of 0.6 µM of NVP-BEZ235 for 24, 48 and 72 hrs, followed by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. The percentage of absolute DNA fragmentation is shown as readout of apoptosis. <b>E</b> 24, 48 and 72 hrs after treatment with 0.6 µM NVP-BEZ235 total cell numbers of treated and untreated cells were counted. <b>F</b> Cell cycle distribution (untreated control and samples treated with 0.6 µM of NVP-BEZ235) was determined after indicated times by FACS analysis of propidium iodide-stained nuclei. <b>G</b> Either untreated controls, or cells treated for 12 and 24 hrs with 0.6 µM NVP-BEZ235 were stained for Ki67 protein expression and evaluated by immunofluorescent microscopy. In A–C and F a representative result of two independent experiments is depicted, while in D and E mean+s.e.m. values of at least three independent experiments carried out in triplicate are shown. Shown in F is the mean of three independent experiments carried out in triplicate, in G the mean+SD of three independent experiments. Statistical analysis was carried out by two-sided Student's <i>t</i>-test; * P-value <0.01; ** P-value <0.001; # P-value <0.0001.