Figure 2.

Rapamycin impairs the p53 response to nucleolar stress. (A) U2OS cells growing on coverslips in 6-well plates were exposed to mycophenolic acid (MPA, 10 μM), 5-fluorouracil (5-FU, 25 μM), doxorubicin (Dox, 1μM), or Act D (5 nM) overnight. Cells were immunostained for nucleolin (red), p53 (green) and counterstained with DAPI to visualize the nuclei (blue) in the upper panel. Objective 100x. In the lower panel 5-FU treated cells were stained for NPM1/B23 and fibrillarin. Objective 63x. (B) U2OS cells were pre-treated with rapamycin (100 nM) or DMSO (control) followed by addition of 5-FU (25 μM), MPA (10 μM) or Dox (1 μM). Protein extracts were made from the cells. Levels of p53 and p21 proteins were determined by immunoblotting. β-actin served as a loading control. (C) As in panel B but U343MGa Cl2:6 cells were used instead. (D) U2OS cells were treated with DMSO (control), 5nM Act D, 25 μM 5-FU, or 1 μM DOX alone or in combination with rapamycin (100 nM as simultaneous treatment) for a total of 18 hours and then pulsed with BrdU for another 6 hours. Coverslips were fixed and immunostained for BrdU incorporation. Data represent a triplicate experiment with at least 200 cells scored per experiment and treatment combination. Shown is percent BrdU positive cells (mean ± SD, *p < 0.05, n.s. – non-significant).