Fig. 2.
Cell cycle-related effects of STs. (A) Effect of STs on S-phase progression. NIH 3T3 cells expressing control vector (CON), POLST, or SVST, as obtained by retroviral infection, were grown overnight in medium containing 0.2% serum. Cells were then incubated in medium containing BrdU for 1 h and subjected to immunofluorescence. Percent cells undergoing S phase (% BrdU +) is measured by BrdU incorporation compared to the total number of cells, as measured by DAPI-stained nuclei. (B) After retroviral infection C2C12 myoblast cell pools were isolated that express GFP, POLST, or SVST. These respective cells were grown to around 60% confluence, and fresh medium containing 10% FCS was added to the cells at this point for 4 hours. Cell lysates were subsequently collected and analyzed by Western blotting for cyclin D expression using actin as a control. (C) Effect of STs on the cyclin D1 promoter. NIH 3T3 cells were cotransfected with cyclin D-luc reporter, small T antigens, or control vector and pCMV-β-Gal. The luciferase activity was normalized using β-galactosidase activity, and results are reported as the number of relative light units (RLU) × 10−3/β-galactosidase activity unit.