Figure 2.

PI3K/mTOR signaling inhibition induces cell death in NOTCH1^MUT but not NOTCH1^WT head and neck squamous cell carcinoma (HNSCC) cell lines in vitro. A. Annexin V/propidium iodide or BrDU-terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assays were used to measure apoptosis in HNSCC cells with the indicated NOTCH1 and PIK3CA mutations, which were treated with 50 nM GSK2126458 (GSK212) for 24 or 48 hours, respectively. Values are the means ± standard deviations of three independent experiments. *p<0.05, unpaired two-tailed Student t-test. B. Western blotting of cleaved PARP (Cl-PARP) and cleaved caspase 3 (Cl-Caspase 3) levels in mutant and wild type cells after GSK212 treatment for 48 hours. C. NOTCH1^MUT and NOTCH1^WT HNSCC cells were plated sparsely and treated with GSK212 at the indicated concentrations for 48 hours. At 14-21 days after treatment, the cells were fixed and stained, images were captured using the GelCount Tumour Colony Counter, and colony numbers and the colorimetric intensity of the colonies were determined by measuring the optical density (OD) at 570 nm. Values are the means ± standard deviations of three independent experiments. *p<0.001, unpaired two-tailed Student t-test. D. Three NOTCH1^WT lines (black text and bars) and three NOTCH1^MUT lines (red text and bars) were treated with increasing concentrations of GSK212 for 4 hours, and levels of PI3K/mTOR pathway proteins were measured by Western blot analysis.