Figure 6.

Silencing of RPL11 mimics the effect of rapamycin on the p53/21 response to nucleolar stress. (A) Combined siRNA/Act D experiment conducted in a sequential fashion. At zero time point U2OS cells were transfected with siRNA directed against RPL11 or siCtrl (final concentration 20 nM). After 18 hours the cells were treated with Act D (5 nM) and some of the samples as indicated were at this time transfected with siRNA targeting MDM2 (siMDM2). After an additional 18 hours of incubation the cells were harvested and extracted of proteins and level of MDM2, p21, p53, β-actin and RPL11 determined by immunoblotting (36 hours after time zero). (B) U2OS cells were transfected with siRNA-1 targeting RPL11 or siCtrl overnight. Cells were thereafter treated with nutlin-3 (10 μM) or Act D (5 nM) for an additional 18 hours. The blotting membrane was probed for RPL11, MDM2, p53, p21, and β-actin. (C) Real-time quantitative PCR (qRT-PCR) was employed to measure relative p21 mRNA levels (a.u, arbitrary units). U2OS cells were treated with rapamycin, rapamycin + Act D, or transfected with siRNA targeting RPL11 (siRPL11–1) and total RNA was prepared. Expression of p21 mRNA was normalized to that of GAPDH mRNA for each sample. (D) Similar to C, but U343MGa Cl2:6 cells were used.