Figure 4

hBD-3 expression inL. pneumophila- infected cells requires AP-1 (c-Jun). (A) A549 cells were pre-incubated with the proteasome inhibitor MG-132 [10 μM] or the NF-κB activation inhibitor [10 μM] for 1 h before infected with L. pneumophila 130b for 24 h. Induction of hBD-3 release was measured by ELISA. (B) To elucidate the contribution of the JNK-AP-1 pathway, L. pneumophila mediated activation of JNK was detected time dependent by western blot analysis and (C) A549 cells were pre-incubated with the JNK inhibitor JNK II [10 μM] for 1 h before infected with L. pneumophila 130b for 24 h. Induction of hBD-3 release was measured by ELISA. (D) c-Jun phosphorylation was assessed by western blotting after infection of A549 cells with L. pneumophila 130b at indicated time points. (E) Furthermore A549 cells were infected with L. pneumophila 130b at indicated time points and the binding patterns of Polymerase II (Pol II), and c-Jun to the hBD-3 promoter were analyzed by ChIP. Representative gels or blots out of three independent experiments are shown. ELISA data presented are means ± SEM of three separate experiments. *, p < 0.05 compared with uninfected control cells; #, p < 0.05 infected cells versus infected cells with specific inhibitors.