FIG. 1.

Macrophages exhibit constitutive activation of NF-κB which is inhibited by PDTC and AdIκBα. (A) PDTC inhibits constitutive NF-κB activation in macrophages. RAW 264.7 cells and primary human macrophages differentiated for 7 days were treated with 200 μM PDTC for 6 and 24 h, as indicated. The cells were harvested, and nuclear extracts were prepared and analyzed by EMSA as described in Materials and Methods. Unlabeled oligonucleotide (Unlab. oligo) was added (+) to the indicated lanes. The locations of the NF-κB p65-p50 heterodimers and p50-p50 homodimers are designated by arrows. The results are representative of three experiments. (B) PDTC decreases TNF-α-induced NF-κB transcriptional activity as measured by luciferase expression. RAW 264.7 cells transiently transfected with a NF-κB promoter reporter (3X-WT-Luc) were incubated with the indicated amounts of PDTC for 30 min and then treated with 5 ng of TNF-α/ml for an additional 12 h. NF-κB promoter activation was determined by measuring luciferase activity, which is expressed as RLU per microgram of protein. The data are presented as the means ± standard errors of duplicate cultures and are representative of three independent experiments. (C) The constitutive activation of NF-κB is diminished in primary macrophages infected with AdIκBα but not in those infected with AdGFP. Seven-day human macrophages were infected with AdIκBα or control AdGFP at an MOI of 100 for 6 and 24 h, as indicated. The cells were harvested, and nuclear extracts were analyzed by EMSA as described in Materials and Methods. The data are representative of three independent experiments.