Figure S5.
Intestinal epithelial NF-κB activation status of S. Tm–infected mice correlates with mucosal expression of NF-κB target genes.(A) Representative image of TNF-PLA in the cecal mucosa of LPS-injected mice as described in Fig. 4 F. Dashed line indicates a crypt without epithelial NF-κB activation. Solid line indicates a crypt with epithelial NF-κB activation. Scale bars: 50 µm. (B) LPS concentrations in the cecum lumen of untreated mice and mice that were streptomycin-pretreated and infected with S. Tm for 24 h (n = 3). (C) Quantification of epithelial NF-κB activation in the cecum of TNF-treated mice with or without isoflurane anesthesia at 15 min.p.inj. (n = 3–5). (D) Relative distribution of intestinal epithelial NF-κB activation status of S. Tm–infected mice described in Fig. 5 A, sorted by time of infection (n = 28). (E and F) Transcript levels of Cxcl2 and Zyx in the cecal mucosa of mice described in A and naive p65GFP-FL mice, grouped according to (E) the time point of infection (color code as in A) or (F) the epithelial NF-κB activation status of the respective mice. Expression levels were normalized to Actb and depicted in 2-ΔCT (n = 33). (G–I) Confocal microscopy images of mice infected with S. Tm for 12 h (n = 6). 3D visualizations in Video 1, Video 2, and Video 3. Boxes in overview images indicate insets. Arrowheads indicate p65+ nuclei (G). Asterisks indicate p65− nuclei (H). Arrows indicate S. Tm in LP (G) or in the lumen (H). Dashed line indicates the epithelium (I). Scale bars: 30 µm (overview images) or 10 µm (insets). (J)C3 transcription levels in untreated and TNF-treated (5 ng/ml, 4 h) NF-κBΔIEC small intestinal epithelial organoids depicted as 2-ΔCT. Expression levels were normalized to Actb (n = 5). (K) Gating strategy for analysis of C3-coated S. Tm in the intestinal lumen (Fig. 5 G). Combined data of two (B and C) or five (J) independent experiments or representative images of five independent experiments (G–I). Statistical analysis: Mann-Whitney U test. *, P ≤ 0.05; **, P ≤ 0.01. Black line: median. Each circle represents one mouse (B–F) or one experiment (average, J).

Intestinal epithelial NF-κB activation status of S. Tm–infected mice correlates with mucosal expression of NF-κB target genes. (A ) Representative image of TNF-PLA in the cecal mucosa of LPS-injected mice as described in Fig. 4 F. Dashed line indicates a crypt without epithelial NF-κB activation. Solid line indicates a crypt with epithelial NF-κB activation. Scale bars: 50 µm. (B) LPS concentrations in the cecum lumen of untreated mice and mice that were streptomycin-pretreated and infected with S. Tm for 24 h (n = 3). (C) Quantification of epithelial NF-κB activation in the cecum of TNF-treated mice with or without isoflurane anesthesia at 15 min.p.inj. (n = 3–5). (D) Relative distribution of intestinal epithelial NF-κB activation status of S. Tm–infected mice described in Fig. 5 A, sorted by time of infection (n = 28). (E and F) Transcript levels of Cxcl2 and Zyx in the cecal mucosa of mice described in A and naive p65GFP-FL mice, grouped according to (E) the time point of infection (color code as in A) or (F) the epithelial NF-κB activation status of the respective mice. Expression levels were normalized to Actb and depicted in 2-ΔCT (n = 33). (G–I) Confocal microscopy images of mice infected with S. Tm for 12 h (n = 6). 3D visualizations in Video 1, Video 2, and Video 3. Boxes in overview images indicate insets. Arrowheads indicate p65+ nuclei (G). Asterisks indicate p65− nuclei (H). Arrows indicate S. Tm in LP (G) or in the lumen (H). Dashed line indicates the epithelium (I). Scale bars: 30 µm (overview images) or 10 µm (insets). (J)C3 transcription levels in untreated and TNF-treated (5 ng/ml, 4 h) NF-κBΔIEC small intestinal epithelial organoids depicted as 2-ΔCT. Expression levels were normalized to Actb (n = 5). (K) Gating strategy for analysis of C3-coated S. Tm in the intestinal lumen (Fig. 5 G). Combined data of two (B and C) or five (J) independent experiments or representative images of five independent experiments (G–I). Statistical analysis: Mann-Whitney U test. *, P ≤ 0.05; **, P ≤ 0.01. Black line: median. Each circle represents one mouse (B–F) or one experiment (average, J).

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