FIG. 2.
PTEN selectively represses endogenous RNA Pol III transcription. (A) PTEN expression was induced in stable U87 cell lines. U87-PTEN or U87-PTEN-C124S cells, engineered to induce the expression of PTEN or PTEN-C124S in the presence of DOX, were treated with DOX for 0, 6, or 24 h. Total protein was isolated and subjected to immunoblot analysis using antibodies against PTEN, phospho-Akt (p-Akt), Akt, or β-actin. (B) Induction of PTEN, but not PTEN-C124S, decreases RNA Pol III transcription. Inducible U87-PTEN cells were transfected with the tRNA gene reporter and treated with DOX. Total RNA was isolated, and RNase protection assays were performed. The change in transcription is calculated based on reporter gene activity in untreated cells (0 h). (C) PTEN represses endogenous RNA Pol III transcription. Inducible U87-PTEN cell lines were treated with DOX (24 h), and total RNA was isolated and reverse transcribed. PCR analysis was performed on serial dilutions of cDNAs to quantify the level of transcription of GAPDH mRNA and endogenous precursor transcripts for tRNALeu, tRNATyr, and 7SL RNA. The change in transcription is calculated based on the level of transcripts in untreated cells (0 h). (D) Induction of PTEN, but not PTEN-C124S, decreases RNA Pol III transcription in vitro. Nuclear extracts were prepared from inducible U87-PTEN cell lines treated with DOX (24 h). Transcription assays were carried out using the tRNA gene reporter as a template and 10 or 20 μg of nuclear extract. The change in transcription is calculated based on the level of transcription in untreated cells (0 h). Representative autoradiographs are shown. Graphs represent determinations from at least three biologically independent samples (means ± SE).