Figure 1.

Suppression of NF-κB inhibits inflammatory responses in primary rat synovial fibroblasts. (A) Induction of NF-κB-mediated transcription in vitro. For luciferase assay, cells were transfected with a 3x wt κBLUC reporter vector followed by an overnight incubation in a low serum (0.5% FBS) medium and stimulated for 3 hr with IL-1β (10 ng/ml), lipopolysaccharide (10 μg/ml), and TNFα (10 ng/ml). The specificity of NF-κB activation was determined by assessing the expression of a 3x mut κBLUC vector (data not shown). (B) Induction of NF-κB DNA-binding activity. For EMSA, quiescent synoviocytes were stimulated for 1 hr with lipopolysaccharide (10 μg/ml) and TNFα (10 ng/ml). The nuclear extracts were preincubated with a radiolabeled κB probe and resolved on PAGE. The specificity of NF-κB binding was assessed by including an excessive amount (100:1) of cold wt and mut NF-κB decoys into the binding reactions. Both major NF-κB bands (arrows) contained RelA subunits, as determined by the supershift by a RelA (p65) Ab (data not shown). (C) AdsrIκBα expression inhibits NF-κB activation in vitro. Cells were transduced with indicated Ad vectors and 2 days later stimulated with TNFα (100 ng/ml) for 30 min. NF-κB DNA-binding activity was determined by EMSA as in B. (D) Suppression of NF-κB inhibits inflammatory responses in vitro. For Northern blotting, cells were transduced as indicated in the legend to C and stimulated with IL-1β (10 ng/ml) and TNFα (10 ng/ml). Total RNA was analyzed by sequentially hybridizing the blot with indicated radiolabeled probes.