H₂O₂ induces EBV reactivation.

<p>(A) H₂O₂ increased intracellular ROS levels in a dose-dependent manner. TW01 and NA cells were treated with various concentrations of H₂O₂ for 24 h. Cells were then stained with the ROS-sensitive probe dihydroethidium (DHE) for 1 h and their fluorescence levels were measured by flow cytometry. (B) H₂O₂ induced EBV reactivation in a dose-dependent manner. NA cells were treated with various concentrations of H₂O₂ for 72 h. Cell lysates were harvested and subjected to immunoblotting analysis for detection of the expression of the EBV lytic proteins, including Rta, Zta, EA-D, and cellular β-actin was used as an internal control. (C) ROS scavengers reduced H₂O₂-induced ROS production. TW01 and NA cells were pretreated with NAC (1mM), catalase (1000 unit/ml) or reduced glutathione (1mM) for 1 h and then treated with MNNG (1μg/ml) for 24 h. Cells were then stained with the ROS-sensitive probe dihydroethidium (DHE) for 1 h and their fluorescence levels were measured by flow cytometry. (D) ROS scavengers inhibited H₂O₂-induced EBV reactivation. NA cells were pretreated with NAC (1mM), catalase (1000 unit/ml) or reduced glutathione (1mM) for 1 h and then treated with MNNG (1μg/ml) for 72 h. Cell lysates were harvested and subjected to immunoblotting for detection of the expression of the lytic EBV proteins. (E) The activity of Rta promoter was induced by H₂O₂, and the induction effect was inhibited by ROS scavengers. Luciferase activities were measured in TW01 cells transfected with the reporter plasmids of Rp or Zp. The cells were pretreatment with ROS scavengers for 1 h, followed by treating with H₂O₂ (500 μM) for another 24 h, and harvested for luciferase activity assay. *: <i>p</i><0.05, **: <i>p</i><0.01, compared to mock treatment of the same cell line or on the same promoter; +: <i>p</i><0.05, ++: <i>p</i><0.01, compared to H₂O₂ (500 μM) treatment of the same cell line or the same promoter.