CD11c⁺ cells induce local epithelial NF-κB activation via TNF. Mice were i.v. injected with LPS and cecal explants imaged at 1 h.p.inj. by two-photon microscopy (representative image and quantification) if not indicated otherwise. (A) Cecum mucosa from p65^GFP-FLxTlr4^−/− mice reconstituted with a 1:10 mix of ActRFP (10%, Tlr4^+/+) and p65^GFP-FLxTlr4^−/− (90%) BM. Analysis of RFP⁺ cells within an epithelial NF-κB activation zone (see Fig. S2 D, n = 10–18). (B–D) Cecal epithelium NF-κB activation of the indicated BMCs or p65^GFP-FL mice pretreated with isotype control/anti-TNF antibody or i.v. injected with TNF and analyzed at the indicated time points (n = 5 or 6). (E) TNF-treated small-intestinal epithelial organoids. Representative image and quantification of NF-κB activation kinetics with 5, 50, or 500 ng/ml TNF (n = 9–17). Lines connect data points from the same organoid. Red dashed line: 50% activation threshold. Black dotted line: no change. (F) Representative images of the cecal epithelium and quantification of epithelial NF-κB activation of p65^GFP-FLxTlr4^−/− mice reconstituted with a 1:20 mix of CD11c-DTR and TNFa^−/− BM, pretreated with DTX (n = 5–8). (B–D and F) Black line: median. *, P ≤ 0.05; **, P ≤ 0.01 by Mann–Whitney U test. Each circle represents one mouse or one organoid (E). Combined data of two (A and B), three (D and E), four (F), or six (C) independent experiments. Scale bars: 50 µm.