Fig 2: Rescue of established colitis by Adv-FAM3D in Fam3D-/- mice.a Protocol for intrarectal infusion of adenovirus. b, c Changes in body weight (%) and colon length (cm). d Colons examined by H&E staining. Scale bar = 100 µm. Data is representative of 1 experiment repeated 2 independent times. Data is presented as the mean ± SEM. Statistical significance was determined by unpaired, 2-tailed Student’s t test.

Fig 3: Constitutive expression of Fam3D in mouse gastrointestinal tract and loss of Fam3D leads to spontaneous colitis.a Fam3D mRNA expression in normal mouse tissues measured by real-time PCR. MLN: Mesenteric lymph nodes. b Mouse Fam3D protein expression in normal tissues measured by Western blot. Tissues from Fam3D-/- mice were used as a negative control. c Representative immunohistochemical staining for Fam3D in mouse colon. Brown color indicates Fam3D. Scale bar = 20 µm; Insert scale bar = 10 µm. d Fam3D mRNA expression in colon fragments measured by real-time PCR. WC: whole colon. cEP: colonic epithelial cells. cLP: colonic lamina propria cells. cM: colonic muscles. e Mouse Fam3D protein expression in colon fragments measured by Western blot. WC: whole colon. cEP: colonic epithelial cells. cLP: colonic lamina propria cells. cM: colonic muscles. f Distal sections of colons obtained from 4 WT or Fam3D-/- mice. H&E sections showing the length of colonic crypts (11 to 20 crypts from each colon). n = 4 in each group. Scale bar = 100 µm; Insert scale bar = 50 µm. g Representative immunochemical staining of Ki67 in the distal sections of the colon from WT or Fam3D-/- mice. n = 4. Data is presented as the mean ± SEM. Scale bar = 100 µm; Insert scale bar = 50 µm. h F4/80-positive macrophages (green, left panel), CD3-positive lymphocytes (red, middle panel) or B220-positive lymphocytes (green, right panel) infiltrating the colon of WT and Fam3D-/- mice in homeostatic state. Scale bar = 50 µm. Positive cells in 10 high-powered microscopic fields (HPF) were counted in six sections from four independent mice from each group. Data is presented as the mean ± SEM per HPF. i The expression of proinflammatory cytokine genes including Il-1, Cxcl1, Cxcl2, Tnfa, and Ccl2 examined by real-time PCR, n = 7. j, k H&E and PAS/Alcian blue staining of samples from 1-year-old mice. Data is representative of one experiment repeated three independent times. Data is presented as the mean ± SEM. Statistical significance was determined by unpaired, two-tailed Student’s t test.

Fig 4: Increased severity of DSS-induced chronic colitis and AOM/DSS-induced carcinogenesis in Fam3D-/- mice.WT (n = 10) and Fam3D-/- mice (n = 9) were given 1.5% DSS in drinking water for 4 days followed by normal water for 2 weeks for 5 cycles. On week 15, mice were euthanized and colons were harvested. a Survival curves of mice. b, c Changes in body weight (%) and colon length (cm). d The number of tumors in the colon of WT and Fam3D-/- mice. White arrow: tumors. e Tumors in the colon examined by H&E staining. Scale bar = 50 µm. NF: neoplastic foci; IL: infiltrated leukocytes. Mice were given 12.5 kg g-1 AOM and followed by 1.5% DSS in drinking water for 3 days. Mice were then given normal water for 2 weeks. The treatment was repeated 3 cycles. f–i Survival curves of mice, body weight loss, colon length and tumor numbers. j–k Tumors developed in the colon of WT and Fam3D-/- mice examined by H&E staining (j). Scale bar = 100 µm (left panels); Inset scale bar = 50 µm (middle panels) and 20 µm (right panels). IG: invasive gland; mm: muscularis mucosa; asterisk: atypical glandular architecture. Dual immunofluorescent staining of ß-catenin (green) and Ki67 (red) in colon tumors of WT and Fam3D-/- mice (k). Scale bar = 50 µm. Data is representative of one experiment repeated two independent times. Data is presented as the mean ± SEM. Statistical significance was determined by unpaired, two-tailed Student’s t test.

Fig 5: Transfer of Fam3D−/− mouse phenotype to WT mice.Mice were co-housed for 4 weeks and then separated for 3 months to examine the divergence of the microbiota. a Principal-component analysis of Bray-Curtis visualized at different time points. n = 8 for each group. b Representative crypt length after 4-week cohousing without DSS treatment. c, d Body weight and colon length on day 7 of DSS treatment. e Histological changes examined by H&E. Scale bar = 50 μm. Mice were treated with 2.5% DSS before and after cohousing at 3 and 5 days. Fecal bacterial DNA was isolated from uncohoused and cohoused WT and Fam3D−/− mice. Data is representative of 1 experiment repeated 2 independent times. f Principal-component analysis of the Jaccard emperor visualized at different time points. Data is presented as the mean ± SEM. Statistical significance was determined by unpaired, 2-tailed Student’s t test.