Figure 4.

(A) Induction of PTEN and treatment with LY294002 affect a similar set of transcriptional targets. U87MG cell lines carrying inducible wild-type (U87.PTEN.WT) or mutant (U87.PTEN.G129R) PTEN were induced with 0.5 μM muristerone A for 36 h or treated with LY294002 for the same amount of time. mRNAs were isolated, converted to double-stranded cDNA. cDNAs were amplified to generate HCRs, labeled with fluorescent dyes, and hybridized to human cDNA microarrays carrying 14,000 probes. The normalized fluorescent ratios for individual array features were computed and then graphed. Scatter plots depict comparison of normalized ratios: U87.PTEN.WT treated (+) or untreated (−) with muristerone A versus the same cells treated or untreated with LY294002 (Left); U87.PTEN.G129R treated or untreated with muristerone A versus the same cells treated or untreated with LY294002 (Center); U87.PTEN.WT treated or untreated with LY294002 versus U87.PTEN.G129R treated or untreated with LY294002 (Right). Values on the axes are positive for ratios greater than 1 or negative reciprocals for ratios less than 1. The positive and negative signs represent induction or repression of gene expression, respectively. (B) RNase protection of HMG CoA reductase and HMG CoA synthase transcripts. U87MG cells carrying inducible PTEN were either induced with muristerone (lanes 1 and 4) or uninduced (lanes 2 and 5). Control (c) represents reactions carried out without total RNA (lanes 3 and 6). Total RNA was isolated and subjected to RNase protection assay by using anti-sense ³²P-labeled probes representing the genes shown above the panel. Protected probes were purified and separated on a polyacrylamide gel as described in Sambrook et al. (21). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA was used as a control to check integrity of mRNAs in the sample and to normalize gene-specific signal.