Fig. 5.

Analysis of cellular PAR after H₂O₂ exposure and following micro-irradiation. (A) Total cellular PAR levels were analyzed using the Trevigen PARP in vivo Pharmacodynamic Assay kit as described in Section 2.7. Cells were treated for 20 min at 4 °C with 0.5 – 2 mM H₂O₂ and allowed to repair for 10 min at 37 °C prior to analysis. Plotted are PAR values in H₂O₂-treated cells relative to untreated control cells of the same cell type, mean values from 3 experiments ± SEM. The control PAR values are 677 ± 56 and 337 ± 54 pg/ml in XC5 and XRE8, respectively. In XRE8, PAR values at all H₂O₂ concentrations were significantly higher than in control cells, whereas in XC5 there was no significant difference between control and treated (P<0.02). (B) Time course of PAR synthesis in XC5 cells (wild-type XRCC1) and in XRE8 cells (C12A XRCC1) at UV micro-irradiation damaged sites at the repair times specified (1–7 min). Representative images of PAR immunostaining at 1 min can be found in Figure 1A. Anti-PAR antibody with Alexa 647 conjugated anti-chicken secondary antibody were used for immunofluorescence analysis as described in section 2.2. At least 5 cells were analyzed at each time point, error bars represent SEM. The asterisk denotes statistical significance (P<0.05) between the two cell types at 6 min.