MATERIALS AND METHODS Cell cultures Cells were cultured at 37 C in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 9% fetal calf serum and penicillin-streptomycin (90 units/ml) in an atmos- phere containing 5% CO2. The cell lines used in this study were human alveolar basal epithelial cell line A549 and the XRCC1-deﬁcient Chinese hamster ovary (CHO) cell line EM9, the latter stably transfected with either empty vector (EM9-V) or his-tagged XRCC1 gene (EM9-XH) as described in (9). siRNA transfection About 1 106 cells were seeded in a 75 cm2 ﬂask and cultured for 24 h before transfection with siRNA. Transfection (10 nM siRNA) was performed using INTERFERinTM (PolyPlus Transfection) according to the vendor’s protocol. The siRNA was purchased from MWG Biotech AG with the oligosequences: PARP1; 50-AAGCCAUGGUGGAGUAUGATT-30 and MPG; 50-AAGAAGCAGCGACCAGCUAGA-30. Repair assay Cells were seeded in 24-well plates, at a density of 5 104 cells per well, and cultured for 24 h before labelling the DNA with 3H-TdR (7.1 kBq/ml) for 24 h. To ensure a low background of SSBs, the cells were washed twice with HBSS++ and incubated in fresh DMEM for 1 h. Treatment with hydrogen peroxide (H2O2) or dimethyl sulfate (DMS) (diluted in HBSS++) was performed for 15 min on ice and was terminated by washing the cells with ice cold HBSS++ twice. Although BER is not com- pletely abolished, the incision rate is reduced 20-fold by a temperature shift from 37 C to 4 C (10). Pre-warmed DMEM was added to the cells, which were then incubated at 37 C for various time intervals to monitor the repair. Treatment with DNA damaging agent and the follow- ing steps were performed in the presence of PARP inhibi- tor 1,8-napthalimide (50 mM) in speciﬁed samples (11). In order to study remaining damages, the pre-warmed DMEM was replaced by pre-warmed DMEM contain- ing 1,8- napthalimide (50 mM), as indicated, 2 h after terminating the treatment with DMS. In the ADU (alkaline DNA unwinding) assay the cells were washed twice with ice cold 0.15 M NaCl and lysed in the DNA unwinding solution (0.15 M NaCl, 30 mM NaOH) on ice, with the exception of the experiments done with A549 cells and the DMS dose-curve, in which the unwinding was performed at room temperature. The unwinding of DNA was continued for 30 min in darkness and was subsequently neutralized by the addition of 20 mM NaH2PO4. The DNA was then fragmented by son- ication (15 sec, Branson soniﬁer B-12, with micro tip), after which SDS (ﬁnal concentration of 0.24 %) was added. The samples were kept frozen for at least 12 h at 20 C. Separation of double-stranded (ds) and single- stranded (ss) DNA was performed on hydroxyl apatite columns, kept at 60 C, as described earlier (12). Brieﬂy, cell lysates were diluted with water (dilution factor 0.4) Nucleic Acids Research, 2011, Vol. 39, No. 8 3167 and 1 ml of each sample was loaded on to a column. The bound DNA was washed with sodium phosphate (10 mM) and ssDNA was eluted with 4.25 ml potassium phosphate at a concentration of 0.1 M, where after dsDNA was eluted with 4.25 ml potassium phosphate at a concentration of 0.25 M. The number of SSBs per cell was calculated from the ratio of ssDNA to dsDNA by correlation with a standard curve obtained by g-irradiation (12–14). Survival assay Cells were seeded in 10 cm Petri dishes 6–8 h prior to treat- mwaesntp. eErfxoprmoseudrefotro 1h5ymdirnogoenn icpeerionxHidBeS(SH+2+O, 2a)ftoerr wDhMicSh the cells were washed and DMEM was added. Exposure to 1,8-napthalimide was performed by the addition of inhibitor to the cellular growth medium 6 h after seeding the cells. After 24 h the medium was replaced with fresh DMEM. The plates were incubated at 37 C under 5% CO2. After 7–10 days, the colonies were ﬁxed, stained (methylene blue dissolved in methanol, 4 g/l) and counted. Calculations are based on the mean value of two replicas per treatment in three separate experiments. Western blot analysis Cells were trypsinized and washed with HBSS + before the addition of protein lysis buffer (1 protease inhibitor cocktail Roche, 1 % NP-40, 150 mM NaCl, 0.1 M Tris pH 8.0). Whole cell lysates were prepared by 30 min lysis on ice with vortexing every 5 min followed by centrifugation, where the pellet was discarded. The protein concentration was determined by the Bradford assay (Hercules, CA) ac- cording to the vendor’s protocol. The lysates (75 mg protein) were resolved by 4–12% NuPAGE Bis-Tris Gel (Invitrogen) for subsequent western blot analysis using antibodies against the following proteins diluted in 5 % milk (in PBS) as indicated: b-tubulin (1:500), PARP1 (1:500) and MPG (HPA006531, Atlas Antibodies AB) (1:500). The gel was transferred to a nitrocellulose membrane and immunoblotted with primary antibody. The secondary antibody (anti-mouse or anti-rabbit) IgG conjugated to horseradish peroxidase was used for detec- tion of the proteins by an ECL western blot detection system (Thermo scientiﬁc). RESULTS Distinct roles of XRCC1 and PARP inhibition in SSB repair It is well established that both PARP1 and XRCC1 are involved in DNA SSB repair (15,16), and that poly(ADPribose) synthesis is required for the recruitment of XRCC1 to sites of oxidative DNA damage (3). To conﬁrm and further investigate the functions of these proteins in SSB repair, we determined the survival and SSB repair capacity in response to hydrogen peroxide in XRCC1 defective EM9 cells stably transfected with empty vector (EM9-V) or wild-type XRCC1 (EM9-XH) in the