Figure 3.

Synthetic mTOR inhibitors blunt the p53 response to nucleolar stress. (A) U2OS cells were treated overnight with temsirolimus (100 nM), PP242 (200 nM), LY294.002 (500 nM) or rapamycin (100 nM) and thereafter fixed and stained for nucleolin (red) and p53 (green). The DMSO vehicle was used as a control. Cells were counterstained with DAPI to visualize the nuclei. Objective 100x. (B) Cultures of U2OS cells were pre-treated with rapamycin for 6 hours (duplicate samples) at concentrations between 5 nM and 500 nM as indicated in the figure. One of the sample sets was then exposed to medium containing Act D (5 nM) and all samples were incubated for another 18 hours. Relative levels of p53, p21 and p-S6K1 (Thr389) were determined by immunoblotting. (C) Similar to B, but U343MGa Cl2:6 cells were used instead. (D) Cultures of U2OS cells were treated with temsirolimus (duplicate samples) at concentrations between 50 nM and 10 μM as indicated for 6 hours. The second set of temsiroliumus treated samples was then also given Act D (5 nM) for the following 18 hours while the other set was exposed to DMSO (control vehicle). Protein extracts were made from each sample and the relative levels of p53, p21, total S6K1, and p-S6K1 (Thr389) were monitored. (E) Similar to panel B, but PP242 was used instead of temsirolimus. (F) U2OS cells were treated with Act D (5 nM) or nutlin-3 (10 μM) alone or in combinations with temsirolimus (100 nM), LY294.002 (500 nM) or PP242 (200 nM). The cells were pre-treated with the mTOR inhibitors for 6 hours before adding Act D or nutlin-3. Relative levels of p53 and p21 were determined by immunoblotting.