Figure 4. SSB activates DDR and induce inflammation through NF-κB.

(a) Neonatal rat cardiomyocytes (NRCMs) were treated with MMS (0.05 mg ml⁻¹ for 10 min) or vehicle control (Mock) and activation of DDR was assessed by western blotting against phospho- or total ATM, H2AX and p53 at the indicated time point. Western blotting against GAPDH was used as a loading control. (b) NRCMs were treated with MMS (0.05 mg ml⁻¹ for 10 min) or vehicle alone (Mock) for 3 consecutive days and activation of DDR was assessed as described in a. (c) NRCMs were transfected with siRNA against Xrcc1 (siXrcc1) or scrambled negative control oligonucleotide (Scramble). Four days later, activation of DDR was assessed as described in a. (d–f) NRCMs were transfected with siRNA against Xrcc1 and/or Atm. Expression levels of inflammatory cytokines were assessed by real-time PCR (d, n=6 each, technical duplicates). Nuclear translocation of NF-κB was assessed by immunofluorescence (e, green). The nuclei of the cells were counterstained with TO-PRO-3 iodide 642/661 (blue). Scale bar, 20 μm. Cells with positive nuclear NF-κB staining were counted (f, n=7, 8, 8, 5, respectively). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer HSD test for (d,f) **P<0.01 between arbitrary two groups. (g) NRCMs were transfected with siRNA against Xrcc1 and treated with NF-κB inhibitor BAY 11-7082 (2 μM) or dimethylsulfoxide as a vehicle control. The expression levels of inflammatory cytokines were analysed by real-time PCR (n=6 each, technical duplicates). Statistical significance was determined by one-way ANOVA followed by the Tukey–Kramer HSD test. **P<0.01 between arbitrary two groups. Column and error bars show mean and s.e.m., respectively.