FIG 9.

Effect of hydrogen peroxide treatment on subcellular localization of HMGB1 and PCV2 replication. (A) H₂O₂ treatment promoted nucleocytoplasmic translocation of HMGB1. PK-15 cells were treated with or without N-acetylcysteine (NAC; 10 mM) before adding 50 μM H₂O₂. Cells were fixed and immunostained with anti-HMGB1 (green) for confocal microscopy. Nuclei were labeled with DAPI (blue). (B) Immunoblotting of HMGB1 in the nuclear and cytoplasmic fractions of PK-15 cells treated with H₂O₂ and NAC. (C) Confocal imaging of PK-15 cells infected by PCV2 with or without 50 μM H₂O₂ treatment after immunostaining with anti-HMGB1 (green) and anti-Cap (red) antibodies. (D) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without H₂O₂ treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (E) Percentages of PCV2-infected cells were calculated from immunofluorescence images as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the H₂O₂-treated cells are shown with nontreated but PCV2-infected cells set at 100%. Bar chart in panel E shows means ± SDs from three independent experiments. **, P < 0.01.