NF-κB activation was correlated with chondrocyte survival mediated by low-concentration sodium nitroprusside (SNP). (a) Activation of NF-κB in 0.1 mM SNP-treated chondrocytes. Chondrocytes were treated with 0.1 mM SNP for 14 hours with or without NF-κB inhibitors and the activation of NF-κB was analyzed by electrophoretic mobility shift assay. Nuclear extracts were prepared from 2 × 106 cells, and 5 μg portions of extracts were used for the binding reaction. Nuclear extracts were incubated in gel binding buffer with radiolabeled consensus double-stranded NF-κB probe, and samples were loaded onto 4% nondenaturing polyacrylamide gel. Protein complexes were identified by autoradiography. Data are representative of three samples from different donors. (b) Effect of the inhibition of NF-κB activation on the protective effect of 0.1 mM SNP on human chondrocytes. Cell death was induced by treating chondrocytes with 1 mM SNP for 24 hours. To inhibit NF-κB, chondrocytes were co-treated with 20 μM Bay 11-7082 or MG132 and 0.1 mM SNP for 14 hours before treating with 1 mM SNP. Cell death was quantitated by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazdium bromide assay. Cell survival in the control culture was set at 100%. Data are the means and standard deviations of triplicate experiments from at least three different donors. * P < 0.05 versus control.
Kim et al. Arthritis Research & Therapy 2005 7:R526 doi:10.1186/ar1705