While the preceding data were obtained from postmitotic differentiated myotubes (>95%), we also determined whether myoblasts would yield comparable results. In myoblasts, the knockdown of PRAS40 decreased global protein synthesis by ~25% under basal conditions (). Despite the decrease in basal protein synthesis in the PRAS40 knockdown cells, the ability of these cells to respond positively or negatively to IGF-I or AICAR, respectively, was unaltered. Contrary to expectations, the decreased protein synthesis observed in PRAS40 knockdown cells under basal conditions was not associated with any difference in phosphorylation state of the mTOR substrates S6K1 and 4E-BP1, compared with the scramble control values () or changes in protein–protein interaction of PRAS40-raptor-eIF3 between the two groups (). In myoblasts, the ability of IGF-I to stimulate T389 phosphorylation of S6K1 and AICAR to increase S792 phosphorylation of raptor did not differ between scrambled and PRAS40 knockdown cells ().
Our data demonstrate the presence of a concomitant delay in proliferation and altered cell cycle in PRAS40 knockdown myoblasts. Because mTOR also regulates autophagy, which in turn plays an important role in cell differentiation (,), we determined the expression of proteins important in regulating autophagy. Although there were no changes in the early markers for autophagy, including Atg 7 and Beclin 1 (), our data indicate that PRAS40 KD decreased the ratio of LC3B-II/LC3B-I ().
Unit 5: DNA/RNA/Cell Cycle/Protein Synthesis
Myoblasts were transfected with either a scramble (control) shRNA or a shRNA targeting PRAS40. Cells were seeded in 10-cm dishes and used at 60% confluence (~24 h postseeding). Cells were trypsinized, washed with DPBS and fixed in cold 70% ethanol overnight at −20°C. Cells were then stained with 100 μg/mL solution of propidium iodide buffer containing 0.1% Triton-X100 and 0.001% DNAse free RNAse at 37°C for 30 min immediately before fluorescence-activated cell sorter (FACS) analysis. A total of 10,000 cells per sample were counted, and cell cycle phase was measured by propidium iodide staining intensity using a BD FACS-Calibur flow cytometer (Becton Dickinson, Bedford, MA, USA) and ModFit software LT Version 3.2 (Verity Software, Topsham, ME, USA).
Translation is a key process in biological lifeforms
C2C12 myoblasts (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s minimum essential medium (DMEM; Invitrogen; Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 μg/mL) (all from Mediatech, Herndon, VA, USA) under 5% CO2 at 37°C. Myoblasts were subcultured and when 100% confluent, the cells were switched to differentiation medium consisting of DMEM with the above antibiotics-antimycotics and 2% horse serum (Hyclone, Logan, UT, USA) to promote myoblast fusion and differentiation to myotubes. Cells were differentiated for 6 d before experimental manipulation. Myotubes were provided with fresh differentiation medium on day 6 and experiments were performed on day 7. To simulate basal mTOR activity, experiments measuring protein synthesis and the phosphorylation of mTOR substrates were performed with serum-free DMEM without antibiotics-antimycotics. 5-Aminoimidazol-4-carboximide ribonucleoside (AICAR; Toronto Research Chemicals, Ontario, Canada), when present, was added at a final concentration of 2 mmol/L for 8 h. Insulinlike growth factor (IGF)-I, when present, was used at final concentration of 100 ng/mL for the last 20 min of the experiment. These doses maximally suppress and activate protein synthesis in C2C12 cells, respectively (,).