Figure 4.

H₂O₂ triggers JWA translocation into the nucleus and co-localization with the components of BER complex on DNA damage sites. (A) NIH-3T3 cells were treated with or without 10 mM H₂O₂ for 20 min and then incubated in H₂O₂-free medium for 10 min, fixed with 4% formaldehyde, permeabilized with 0.01% Triton X-100 and then immunostained with anti-JWA (red) or anti-PAR antibody (green). (B) Cells were treated with H₂O₂ as in (A) and stained with anti-JWA (red) or anti-XRCC1 antibody (Green). (C) NIH-3T3 cells were transiently transfected with a GFP-JWA or an RFP-XRCC1 plasmid. After 48 h, the transfected cells were split, grown on coverslips, exposed to H₂O₂ as in (A). Representative images were photographed and colored using a Zeiss LSM 510 confocal microscope system. (D) The intracellular distribution of JWA and the components of BER complex during DNA repairing after H₂O₂ exposure. NIH-3T3 cells were treated with 100 μM H₂O₂ for 30 min and further cultured in H₂O₂-free medium to allow for DNA repair. Cytoplasmic and nuclear extracts were prepared, and western blotting were employed to detect the expression of XRCC1, LigIII, PARP-1 and JWA. Aldolase and histone H1 were used as the cytoplasmic and nuclear loading controls, respectively. The experiments were repeated twice with similar results. (E) XRCC1 does not affect the expression of JWA. NIH-3T3 cells were transfected with a control siRNA pool or a XRCC1 siRNA pool to knockdown endogenous XRCC1. After 72 h, the transfected cells were treated with or without 100 μM H₂O₂ for 30 min. Whole-cell lysates were collected for detection of target proteins, including XRCC1 and JWA by immunblotting. β-Actin was used for the protein loading control. (F) XRCC1 induced subcellular redistribution of JWA and LigIII. The treatments were the same as in (E). Western blot analysis was performed to analyze XRCC1, LigIII and JWA in the cytoplasmic and nuclear extracts. Aldolase and histone H1 were used as the cytoplasmic and nuclear loading controls, respectively.