Figures & data
Figure 1. Protein Expression Levels of mTOR Mutations.HEK293T cells stably expressing mTOR mutants (H2189Y, P2213S and S2215Y) were constructed by TALEN. After nutrient starvation, wild type or mutated HEK293T cells were lysed.The phosphorylation levels of mTOR, p70S6K, AKT and 4EBP1 were examined by Western blot (A).Results in (B-E) were quantified by measuring the relative intensity of phosphorylated protein bands to corresponding total protein bands with the method of mean ± SD of 3 scans.
Figure 2. mTOR kinase activity of mTOR mutants.HEK293T cells stably expressing mTOR mutants (H2189Y, P2213S and S2215Y) were constructed by TALEN. After nutrient starvation, wild type or mutated HEK293T cells were lysed, and the activation of mTOR kinase were examined by K-LISA™ mTOR (Recombinant) Activity Kit.
Figure 3. Protein Expression Levels of mTOR mutations after PI3K-AKT-mTOR inhibitors treatment. HEK293T cells stably expressing mTOR mutants (H2189Y, P2213S and S2215Y) were constructed by TALEN. After nutrient starvation, wild type or mutated HEK293T cells were treated with indicated inhibitors or vehicle for 24 hours. The activation of indicated molecules was examined by Western blot.
Figure 4. Sensitivity of gain-of-function mTOR mutations to PI3K-AKT-mTOR inhibitors. HEK293T cells stably expressing mTOR mutants (H2189Y, P2213S and S2215Y) were constructed by TALEN. After nutrient starvation, wild type or mutated HEK293T cells were treated with indicated inhibitors or vehicle.The proliferation of HEK293T cells was evaluated by CellTiter-Glo® Luminescent Cell Viability Assay, and the results were presented as mean ± SD of 3 independent experiments.
Figure 5. Sensitivity of xenograft models to inhibitors in vivo. When the tumor size reached approximately 200 mm3, mice were treated with buffer control or inhibitors daily. Tumor volume was evaluated as % of the tumor volume on day 0 and presented as mean ± SD. The data are representative of these independent experiments.
