Fig. 5.

Effect of STs on cell survival. NIH 3T3 cells expressing small T antigens were obtained by retroviral infection. (A) Cell survival following retroviral infection. Cells were infected with retrovirus containing only the puromycin resistance gene (pBABE-puro) as a control (CON) expressing POLST (pBABEpuro-POLST) or SVST (pBABEpuro-SVST). After selection in puromycin (2.5 μg/ml) for about 7 days, cells were fixed with ethanol, stained with 0.2% crystal violet, and washed (78). (B) Expression of STs was confirmed by GFP expression as seen in fluorescence microscopy, and the morphology of the cells expressing POLST indicated cell death. Nuclei were stained by DAPI and observed by fluorescence microscopy. (C) POLST induced apoptosis (APOP) as seen by FACS analysis. A sub-G₁ peak was seen in the cell cycle histogram of POLST-expressing cells but not in control (CON) or SVST-expressing cells. FACS analysis showed that 37% of the infected cells with POLST had sub-G₁ DNA content. (D) Apoptosis in POLST-expressing cells as indicated by DNA fragmentation. DNA was extracted from ST-expressing NIH 3T3 cells, separated on a 2% agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator.