FIG 4.
EBV Rta expression is accompanied by ROS production and increased binding activity of DNMT1/DNMT3A in target cellular promoters. (A) Cellular levels of reactive oxygen species (ROS) were measured by using dihydroethidium (DHE) staining (200× magnifications). 293TetLuc and 293TetER cells were incubated with culture medium (Ctrl), 500 μM H2O2, or 50 ng/ml doxycycline (Dox) for 6 h, followed by Hoechst 33258 or DHE staining. Percentages of DHE-positive cells, determined by counting ≥300 cells (200× magnifications) for each treatment, are summarized on the right. Error bars indicate the means ± SD from four independent experiments. (B) Rta-mediated MYC, CCND1, and JUN downregulation was blocked by N-acetyl-l-cysteine (NAC). 293TetLuc and 293TetER cells were pretreated with 0.5 or 1 mM NAC for 1 h followed by 500 μM H2O2 or 50 ng/ml Dox treatment for 6 h. Cellular RNAs were extracted and analyzed by real-time RT-PCR. Data are presented as the means ± SD from four independent experiments. The statistical significances of untreated and drug-treated cells were evaluated by Student's t test: ***, P < 0.005; **, P < 0.01; *, P < 0.05. (C) Binding activities of DNMT1 and DNMT3A were increased in Rta-expressed cells. Untreated (Ctrl), H2O2-treated, and Dox-treated (6 h) 293TetLuc and 293TetER cells were subjected to ChIP assays using specific antibodies against DNMT1 and DNMT3A. Normal rabbit IgG was used as a control to estimate nonspecific binding (Fig. S1B). In the real-time PCR assays, data are shown as percentages of input using the ΔCT method. Error bars represent the means ± SD from four independent experiments. Student's t test was used to assess statistical significance: ***, P < 0.005; **, P < 0.01; *, P < 0.05.