FIGURE 7.

Induction of DNA damage response and cell cycle arrest at S phase by IBV nsp13. a, induction of γH2AX by overexpression of IBV nsp13 is shown. H1299 and HeLa cells were transfected with pXJ-Myc and pXJ-Myc-nsp13, respectively, using Lipofectamine 2000 Transfection kit. Cells were harvested at 48 h post transfection, lysed, and analyzed by Western blot with specific anti-γH2AX antibodies. The same membranes were also probed with anti-actin and anti-Myc antibodies. b, immunofluorescent staining of γH2AX in cells overexpressing Myc-nsp13 is shown. H1299 cells were transfected with pXJ-Myc and pXJ-Myc-nsp13 using the CalPhos Mammalian Transfection kit. At 48 h post-transfection, cells were fixed and double immuno-stained with mouse anti-Myc and rabbit anti-γH2AX antibodies. c, induction of cell cycle arrest at S phase by overexpression of IBV nsp13 is shown. H1299 cells were transfected with pXJ-Myc and pXJ-Myc-nsp13 and incubated for 40 h. Cells were harvested by trypsin treatment, fixed with 80% ethanol, and stained with mouse anti-Myc antibody and PI for FACS. The DNA contents of cells were measured by PI, and the Myc-nsp13 (+) cells were viewed with anti-Myc antibody. A total of 100.000 cells were counted in each experiment. Results of representative experiments performed in triplicate are shown. FITC, fluorescein isothiocyanate, d, cell cycle profiles in cells overexpressing Myc-nsp13 are shown. Statistical analysis was carried out by t test (p < 0.001, n = 3).