ExoU Activates NF-κB and Increases IL-8/KC Secretion during Pseudomonas aeruginosa Infection
Figure 3
ExoU promotes p65/p50 nuclear translocation and NF-κB-dependent transcriptional activity in P. aeruginosa-infected A549 cells.
In (A), representative EMSAs showing NF-κB nuclear translocation after 2 and 12 hours of P. aeruginosa infection or treatment with culture medium. To evaluate NF-κB inhibition by Bay 11-7082, some cultures were treated with 10 µM Bay 11-7082, 1 hour prior PA103 infection. As a control for non-specific interactions, nuclear extracts from PA103-infected cells were also incubated with a probe mutated in a single nucleotide (Mut). In (B), extracts obtained from PA103-infected cells were supershifted with specific antibodies against NF-κB subunits, p50 and p65. In “Non-infected cells” and “PA103-infected cells” lanes, antibodies were not added. EMSA and supershift assays were performed in triplicate. In (C), the graph shows the luciferase activity (in arbitrary values) of whole-cell lysates obtained from A549 cultures transfected with p6κB-LUC and pRL-CMV plasmids and then infected with P. aeruginosa strains or treated with cultured medium for 24 hours. Data represent means ± SEM of three different assays carried out in quadruplicate. **p<0.01 and ***p<0.001 when the values obtained from PA103ΔexoU-infected cultures and non-infected cultures, respectively, were compared with those obtained from PA103-infected cultures.