<i>Bb</i> and its soluble factors inhibit TNFα-induced NF-κB in epithelial cells.

<p>A. HT29-19A cells were incubated for 30 min with TNFα±<i>Bb</i>-CM and localization of the NF-κB p65 subunit was observed by confocal microscopy. Following exposure to TNFα, p65 (green) was translocated into the nuclei (blue) and co-localization was observed (white spots). <i>Bb</i>-CM inhibited TNFα-induced p65 subunit translocation. n = 3 independent experiments. B. HT29-19A cells were incubated for 4 hours with TNFα±<i>Bb</i>-CM. Electrophoretic migration shift assay (EMSA) was performed following extraction of nuclear proteins. TNFα condition was taken as 100% activation and <i>Bb</i>-CM induced a 40% inhibition of NF-κB activation (n = 2 independent experiments). C. Epithelial cells were incubated with TNFα±<i>Bb</i> at MOI 100. Activation of NF-κB was inhibited by ∼40% with <i>Bb</i> but not with commensal bacteria <i>B.breve</i> ATCC 15698, <i>L.rhamnosus</i> 10863, <i>E.rectale</i> L15. n = 2 independent experiments. D. HT29-19A cells were treated with TNFα±<i>Bb</i>-CM. Total proteins were separated by SDS-PAGE and revealed with antibodies to phospho-IκB-α, IκBα and β-actin. <i>Bb</i>-CM inhibited the TNFα-induced phosphorylation of IκB-α as early as 5 min and IκB-α degradation after 15 min. n = 3 independent experiments.