PDF《Supplementary Figures》

Supplementary Figures Supplementary Figure 1.

A20IEC/myel-KO mice have expanded myeloid cell populations and produce high serum titers of inflammatory cytokines. (a) Spleen weights of wild-type (WT, n=11), A20myel-KO (n=8) and A20IEC/myel-KO (n=15) littermate mice. (b) CD11b+ FACS analysis of splenocytes from wild-type (WT, n=5), A20myel-KO (n=5) and A20IEC/myel-KO (n=10) littermate mice. (c, d) Levels of IL-6 (c) and TNF (d) in serum from wild-type (WT, n=24), A20IEC-KO (n=20), A20myel-KO (n=11) and A20IEC/myel-KO (n=29) littermate mice at the age of

22 weeks. Error bars represent SEM. * p < 0.05 using Student's t- test. Supplementary Figure 2. Ileitis and severe colitis in aged (30-50 weeks old) A20IEC/myel-KO mice. A, crypt loss;

B, distortion of mucosal architecture;

C, mixed inflammatory infiltrate in the lamina propria mucosa and basal plasmacytosis;

D, submucosal accumulation of mononuclear cell infiltrates (mainly plasma cells);

E, lymphoid aggregates;

F, neutrophil infiltration with epithelial erosion;

G, crypt abscesses. Scale bars,

50 ?m. Supplementary Figure 3. Normal intestinal stem cell marker expression in A20IEC/myel-KO mice. Quantitative PCR for intestinal stem cell markers on ileal IEC lysates from

20 week old wild-type (WT, n=4) and A20IEC/myel-KO (n=8) mice. Error bars represent SEM, n.s.: not significant, p >0.05 using Student's t- test Supplementary Figure 4. A20 deficiency does not affect Wnt signalling and gene expression in vitro and in vivo. (a, b) Immunohistochemical staining of ileal sections of wild-type (WT) and A20IEC/myel-KO mice with antibodies specific for β-catenin (a) and Sox-9 (b). Arrows indicate granular Paneth cells (Scale bars, upper two panels:

10 ?m, lower panel:

5 ?m). (c) Quantitative PCR for Cyclin D1 and c-Myc on wild-type (WT, n=3) and A20-/- (n=3) intestinal organoid cultures stimulated in vitro with recombinant Wnt3 (200 ng/ml). Graph is representative of two independent experiments. Error bars represent SEM. Supplementary Figure 5. Paneth cells in A20IEC-KO mice are lost in response to recombinant TNF injection. (a) Immunofluorescent staining of Lysozyme (green) and TUNEL (red) on ileal sections of A20IEC-KO mice. Arrows indicate dying Paneth cells (Scale bars,

5 ?m). (b) Quantitative PCR for LysP and Crypt-1 on IEC ileal lysates from A20IEC-KO mice after injection with

1 ?g recombinant mouse TNF. Supplementary Figure 6. Representative immunofluorescent pictures detecting cleaved caspase-3 in wild-type (WT) and A20-/- gut organoids

4 h after stimulation with vehicle (control), TNF or IFNγ (10 ng/ml). Supplementary Figure 7. A20 expression in mucosal biopsies correlates to primary infliximab responsiveness. Individual microarray normalized log2 expression values for A20 in intestinal mucosa from IBD patients before and after first infliximab treatment compared to controls. Lines between

2 points represent the change in expression before and after treatment for each IBD patient. CDi: Crohn's ileitis, IBDc: colonic inflammatory bowel disease, NR: infliximab non-responders, R: infliximab responders, * p < 0.001: unpaired samples were analyzed with the Mann-Whitney U-test , and paired samples with the Wilcoxon signed-rank test for paired samples. Supplementary Figure 8. Schematic representation of IBD/CAC development in A20IEC/myel- KO mice: A20 deficient intestinal epithelium is hypersensitive to cytotoxic cytokines produced by hyperactive A20 deficient myeloid cells. IEC apoptosis leads to Paneth and goblet cell loss, barrier destabilization and bacterial infiltration. Chronic inflammation and IEC hyperproliferation sensitize for the development of colorectal cancer in aged mice (SI: small intestine). Supplementary Tables Sample Gender Cage Age Genotype QQ Reads Obs Richness Chao1 P1.112 f