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. 2015 Aug 18;5:13252. doi: 10.1038/srep13252

Figure 8. E. coli O104:H4 OMVs induce IL-8 secretion by human IECs via flagellin and LPS.

Figure 8

(a) Caco-2, HCT-8, and HT-29 cells were incubated with OMVs LB226692 (containing 58 ng/ml of Stx2a, 80 ng/ml of flagellin, and 950 ng/ml of LPS) or OMVs C227-11Φcu (containing 80 ng/ml of flagellin and 950 ng/ml of LPS) for 15 min, 1 h, 3 h, and 24 h; IL-8 in cell culture supernatants was quantified by a Single-Analyte IL-8 ELISArray; *P < 0.05 (unpaired Student’s t test) for comparison between OMVs LB226692 and C227-11Φcu. (b) Cells were exposed for 24 h to: OMVs or isolated H4 flagellin (80 ng/ml) in the absence of antibodies; OMVs or flagellin which had been preincubated with anti-H4 antibody; OMVs or flagellin after cell preincubation with anti-TLR5 antibody; OMVs or flagellin which had been preincubated with anti-H4 antibody after cell preincubation with anti-TLR5 antibody. IL-8 secretion was quantified as above; **P < 0.01 or ***P < 0.001 (one-way ANOVA) for comparison between each OMV preparation or flagellin without and after preincubation with each respective antibody combination. (c) IL-8 secretion was quantified in cells incubated for 24 h with OMVs or isolated O104 LPS (950 ng/ml) without polymyxin B (polyB-) or with OMVs or LPS which had been preincubated with polymyxin B (polyB+); *P < 0.05 (one-way ANOVA) for comparison between each OMV preparation or LPS without and after polymyxin B preincubation; **P < 0.01 (one-way ANOVA) for comparison between each OMV preparation and isolated O104 LPS. (d) Cells were incubated for 24 h with 10-fold dilutions of OMVs LB226692 or C227-11Φcu containing the indicated amounts of flagellin and LPS; IL-8 secretion was quantified as above. Data in panels (a–d) are means ± standard deviations from three independent experiments. (e) Detection of TLR5, TLR4 and MD-2 in lysates of Caco-2, HCT-8, and HT-29 cells using immunoblot. Signals were visualised with Chemi Doc XRS imager. Sizes of immunoreactive bands are shown on the right side. Crops of representative immunoblots are shown. Full immunoblots are shown in Supplementary Fig. S10.