Figure 3.
The IRES domain of the p27 5′UTR confers resistance to translational repression by PI3-kinase inhibitor LY294002. (A) Protein synthesis rates of HeLa cells after addition of PI3-kinase inhibitor LY294002. Cells treated with LY294002 or solvent (ethanol) for 0 min, 10 min, 2 h, 5 h, or 24 h were labeled with (35S)-methionine for an additional 30 min. The incorporation of radioactivity into proteins in LY294002 treated cells was plotted as a percentage of the incorporation in solvent-treated cells. (B) Analysis of PI3-kinase inhibition on the translation of monocistronic mRNAs with p27 5′UTR leaders. Transfected HeLa cells were incubated for 24 h with LY294002 or solvent. Luciferase acivities were normalized to luciferase mRNA levels determined in Northern blots. (C,D) Endogenous p27 protein and mRNA levels are unaltered in LY294002-treated cells. HeLa cells were incubated with LY294002 (LY) or the solvent (C). p27 mRNA levels (top) were analyzed by Northern blot of total RNA with probes specific for p27 mRNA and GAPDH mRNA (bottom). The 18S small ribosomal RNA serves as an additional loading control. (D) Protein levels of p27 (top), p21 (middle), or α-tubulin (bottom) were detected by immunoblotting using monoclonal antibodies. (E) Synthesis rates of p27 are unaltered in the presence of LY294002. After 23-h incubation with LY294002 or solvent (control), adherent HeLa cells were pulse-labeled with (35S) methionine and (35S) cysteine for 1 h. Extracts adjusted for equal amounts of protein were boiled and p27 was immunoprecipitated. p27 synthesis was quantified after SDS-PAGE using a PhosphorImager. (Left) The average of six experiments is represented. (Right) Incorporation of radioactivity in total protein was determined by TCA-precipitation of protein extracts of identical protein concentrations from solvent (C) or LY294002 (LY)-treated cells and liquid scintillation counting. Three experiments are represented. (F) Flow cytometry analysis of cell cycle distribution of adherent HeLa cells after 24 h treatment with LY294002 or solvent.