D4522

The concentration of serum DNase I was measured using a commercial ELISA kit according to the manufacturer’s instructions (EIAab, Catalog No: E1127h, Wuhan, China). The optical absorbance was measured at 450 nm in an ELISA reader (Bio-Rad 550; Bio-Rad Laboratories, Tokyo, Japan). Samples were compared to the standard curve and the results were expressed in ng/mL.
Among the above-mentioned patients, we recruited 22 patients in active stage with sufficient serum samples, and randomly selected 30 patients in remission, to detect the DNase I activity. DNase I activity was measured using radial enzyme-diffusion method, as previously described [24 (link)]. To 12ml of 10mM Tris/Ca/Mg buffer, 1.2ml of DNA from calf thymus (D4522, sigma) at 5mg/ml, 3μl ethidium bromide (EB) and 13.2ml melted 2% agarose (Biowest), were added. Then the substrate was poured onto the gel plate. Wells were cut of 1mm diameter, filled with 2μl standards and serum sample, then the plate was incubated at 37°C overnight. The plate was photographed on a UV Transilluminator. The area of dark circles of hydrolysed DNA was scanned by Image-Pro Plus 6.0.

Inhibition of PARP-1 activity was evaluated in a radioisotope-based filter binding assay by Reaction Biology Corporation (Malvern PA) using human recombinant PARP-1 (residues 1-1014, Genbank accession # NM_001618.3, MW = 114.8 kDa) expressed with a C-terminal His-tag in Sf9 insect cells, >80% pure by SDS-PAGE. Polymerization of ³²P-NAD⁺ (10 µM) on core histones (0.01 mg/mL) in buffer (50 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl₂, 0.02% Brij35, 1 mM DTT, 1% DMSO and 20 µg/mL activated DNA (Sigma-Aldrich D4522) was measured after 1 h incubation and washout of remaining free NAD⁺. Each compound was included in a 10-point, 3-fold dilution dose response experiment and an IC₅₀ value derived.