FIG. 3.
Decreasing PTEN expression selectively enhances RNA Pol III transcription. (A) Reducing expression of PTEN enhances RNA Pol III transcription in LN18 cells. LN18 cells were transfected with PTEN siRNA or control mmRNA. (Top) Total protein was isolated, and immunoblot analysis was performed using antibodies against PTEN, phospho-Akt (p-Akt), Akt, or β-actin. (Bottom) Cells were cotransfected with the tRNA reporter gene and PTEN siRNA or mmRNA. Total RNA was isolated, and RNase protection assays were performed. Representative autoradiographs are shown. The change in transcription is calculated based on reporter gene activity in cells transfected with mmRNA. (B) Reducing expression of PTEN enhances RNA Pol III transcription in MCF-7 cells. MCF-7 cells were transfected as described above (A). (Top) Total protein was isolated, and immunoblot analysis was performed using antibodies against PTEN and β-actin. (Bottom) Total RNA was isolated, and RNase protection assays were performed as described above (A). (C) Decreasing PTEN expression selectively enhances endogenous RNA Pol III transcription. LN18 cells were transfected with PTEN siRNA or mmRNA. Total RNA was isolated and reverse transcribed. PCR analysis was performed on serial dilutions of cDNAs to quantify the expression of β-actin mRNA, precursor tRNATyr, precursor tRNALeu, and 7SL RNA. The change in transcription is calculated based on transcript levels in cells transfected with mmRNA. Graphs represent determinations from at least three biologically independent samples (means ± SE).