Resveratrol suppressed AGEs-stimulated NF-κB signaling. Chondrocytes were cultured overnight with serum-free medium and then pretreated with various doses of resveratrol or the solvent (V) for 24 h and then stimulated with 100 μg/ml AGEs for another 24 h. The cells were collected and the nuclear extracts were prepared for determination of the DNA-binding activity of NF-κB by EMSA (a). In (b), the total cell lysates from chondrocytes treated with AGEs for various time points were collected for determination of the levels of IκBα by Western blot. Similar to (B), except that the cells were pretreated or not with resveratrol before the stimulation with 100 μM AGEs for 4 h (c). In (d), similar to (B), the levels of phosphorylated IKKα/β and total IKKα were determined by Western blot using specific antibodies that recognize phosphorylated IKKα/β or unphosphorylated IKKα. In (e), similar to (D), the effects of resveratrol on the levels of phosphorylated IKKα/β and total IKKα induced by AGEs treatment for 30 min were determined. In (f), the cells were co-transfected with 1 μg of the pNF-κB-luciferase reporter plasmid and the internal control plasmid pTK-Renilla-luciferase at a ratio of 100:1. After transfection, the cells were equally distributed for individual conditions and then pretreated or not with different concentrations of resveratrol for 24 h and then treated with 100 μg/ml AGEs for another 24 h. The cells were collected and the total cell lysates were prepared for luciferase activity determinations. The results were shown as fold inductions of luciferase activity as compared to the unstimulated sample. Values are means ± standard deviations (error bars) for three independent experiments. *: P < 0.05 compared to the AGEs-stimulated in the absence of resveratrol treatment.