Figure 5

Mapping the shared epitope (SE)-triggered pro-oxidative signaling pathway. (a) M1 cells were pre-incubated for 20 minutes with the nitric oxide synthase (NOS) inhibitor L-NMMA (500 μM) and exposed to Sepharose bead-immobilized allele-specific 15 mer peptides for 1 hour, and the extent of adenosine-induced protection against oxidative DNA damage was determined. (b) M1 cells were incubated with or without 50 μg/ml of peptide 65–79*0401 or 65–79*0404 in the presence or absence of NOS1 inhibitor TRIM (trifluoromethylphenylimidazole) (250 μM) or the NOS2-selective inhibitor 1400 W (0.2 μM). After overnight incubation, cells were washed, stimulated with 2-chloroadenosine, followed by oxidative challenge with H₂O₂, and the extent of DNA damage was quantified. The concentration of each inhibitor was adjusted to 10-fold higher than its IC₅₀. *p <0.02; **p <10^-5. (c) Dose-response curve of TRIM effect. Cells were incubated overnight with 50 μg/ml of peptide 65–79*0404 in the presence of different concentrations of TRIM. At the end of incubation, cells were collected and adenosine-induced protection against DNA damage was quantified as above. (d) M1 cells were pre-incubated for 10 minutes with the PKG inhibitor KT5823 (1 μM) and subsequently exposed to Sepharose bead-immobilized peptide 65–79*0401. The extent of adenosine-induced protection against oxidative DNA damage was determined. IC₅₀, half inhibitory concentration; L-NMMA, L-N^G-monomethyl arginine citrate; PKG, cGMP-dependent kinase.