Fig 2: 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 3: Altered composition of microbiota in the colon of Fam3D deficient mice.WT and Fam3D−/− mice were pretreated with a cocktail of ABX (vancomycin, imipenem/cilastatin, and neomycin) for 4 weeks prior to administration with 2.5% DSS in drinking water. a Crypt length of mouse colon treated with ABX. b, c Body weight and colon length. d Histological changes examined by H&E. Scale bar = 50 μm. e Representative Alcian blue staining. Scale bar = 50 μm. Data is representative of 1 experiment repeated 2 independent times. f Principal-component analysis of the Jaccard emperor visualized in naïve mice. g Analysis of specific bacterial 16S rDNA to detect commensal diversity in WT and Fam3D−/− mice at the phylum level. h Relative abundance of fecal microbiota at the phylum level confirmed by real-time PCR. i Analysis of specific bacterial 16S rDNA to detect commensal diversity in WT and Fam3D−/− mice at the family level. j Families selected from mice with given genotypes. For microbiome sequencing, n = 5 for each group. For real-time PCR, n = 8 for each group. Data is presented as the mean ± SEM. Statistical significance was determined by unpaired, 2-tailed Student’s t test.

Fig 4: 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 5: Reduced homeostatic molecules and defective mucosal integrity in the colon of Fam3D−/− mice.a Heatmaps of differentially expressed genes enriched in host defense responses. b mRNA expression levels of antibacterial peptides in the colons of WT or Fam3D−/− mice measured by real-time PCR. n = 8. c Reg3γ protein levels measured by immunofluorescent staining. n = 4 for each group. d GSEA analysis on Fam3D−/− vs WT EBSeq log2 FC expression data of colonic epithelial samples. e Immunofluorescent staining for Fam3D and MUC2 in normal colon tissue. Data is representative of one experiment repeated three independent times. f Double staining of Acian blue and PAS. Yellow arrow: Acian blue positive materials; orange arrow: PAS single positive cells; orange star: dilated goblet cells, goblet cells enumerated in each crypt (10–20 crypts from each colon) and pixels of Alcian blue staining areas. Alcian blue staining areas and fluorescence intensity quantified by Image J based on six distal colon sections of four independent mice from each group. g Staining of high iron diamine/Alcian blue, dark brown, and black were sulfomucins, whereas blue staining indicates sialomucins, sulfomucin positive cells enumerated in each crypt (10–20 crypts from each colon) and the ratio of sialomucins to sulfomucins is calculated. Data is presented as the mean ± SEM. Scale bar = 50 μm. h Representative Alcian blue staining of Carnoy’s-fixed colonic sections. The thickness of the inner mucus layer (light blue band between luminal content and the mucosa) was quantified (5–10 fields from each colon) by Image J. n = 8. i Representative dual staining for UEA-I (immunofluorescence, green) and bacteria (fluorescence in situ hybridization, red), the latter using the universal EUB338 probe, on Carnoy’s-fixed colonic sections. n = 8. j PAS/Alcian blue staining of the colons from WT and Fam3D−/− mice with different ages. n = 4. Data is representative of one experiment repeated three independent times. k Quantitation of crypt length of WT and Fam3D−/− mice at different ages. Data is presented as the mean ± SEM. Statistical significance was determined by unpaired, two-tailed Student’s t test.