FIGURE 2.

mTOR activity is required for Pol III transcription. A, MG63 (top), MDA361 (middle), or HEK293 (bottom) cells were treated with vehicle-DMSO, 0.5 μm CCI-779, 0.5 μm WYE-132, 5 μg/ml U0126, 10 μg/ml α-amanitin, or a 100 ng/ml taxol for 3 h. qRT-PCR analysis was used to measure the expression of tRNA^Leu (gray-shaded bars) or tRNA^Tyr (open bars) precursors as described under “Experimental Procedures.” The expression levels of each gene were first normalized with control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data expressed as -fold differences over control untreated samples. The experiment was performed in triplicate. B, MG63 cells were transfected with control (C), mTOR, raptor, or rictor siRNA pools for 72 h as described under “Experimental Procedures.” Left, total lysates were immunoblotted with mTOR, raptor, rictor, P-S6K, P-AKT, and β-actin. Right, expression of precursor tRNA^Leu (shaded bars) or tRNA^Tyr (striped bars) was measured by qRT-PCR. Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. The values represent -fold differences in expression upon siRNA depletion relative to those of the control siRNA. The data shown are representative of three independent experiments. Error bars represent the range around the mean -fold changes as described under “Experimental Procedures.”