NF-κB signal is essential for activation of IL-8 expression by L. pneumophila.(A) Bay 11-7082 and LLnL inhibit IL-8 mRNA expression induced by L. pneumophila. A549 cells were pretreated with Bay 11-7082 (20 μM) and LLnL (20 μM) for 2 h prior to AA100jm infection. They were subsequently infected with L. pneumophila for 6 h. IL-8 mRNA expression on harvested cells was analyzed by RT-PCR. Representative results of three similar experiments in each panel are shown. (B) Functional effects of IκBα, IκBβ and IKKγ dominant interfering mutants and kinase-deficient IKKα, IKKβ and NIK mutants on L. pneumophila-induced activation of the IL-8 promoter. A549 cells were transfected with 40 ng of -1481-luciferase construct and 2 μg of the indicated mutant plasmids or empty vector (pCMV4), and then infected with L. pneumophila (MOI of 100) for 48 h. Open bar represents luciferase activity of empty vector without L. pneumophila infection. All values were first calculated as a fold induction relative to the basal level measured in uninfected cells. Data are mean ± SD values of three independent experiments. *, P < 0.0005 (compared to uninfected cells). **, P < 0.05; #, P < 0.01;##, P < 0.005 (compared to cells transfected with empty vector with further L. pneumophila infection).