Journal: Nature Communications

Article Title: FAM3D is essential for colon homeostasis and host defense against inflammation associated carcinogenesis

doi: 10.1038/s41467-020-19691-z

Figure Lengend Snippet: 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 log 2 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.

Article Snippet: Rat anti-EpCAM antibody (sc-53532), mouse anti-β-catenin antibody (sc-7963), rabbit anti-mouse Mucin 2 (MUC2) antibody (H300) were purchased from Santa Cruz (Dallas, TX).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Double Staining, Fluorescence, Immunofluorescence, Bacteria, In Situ Hybridization, Quantitation Assay, Two Tailed Test

Journal: Mucosal immunology

Article Title: Hypoxia-inducible factor 1 in dendritic cells is crucial for the activation of protective regulatory T cells in murine colitis.

doi: 10.1038/mi.2015.67

Figure Lengend Snippet: Figure 6 Dendritic cell-specific knockout of hypoxia-inducible factor-1a (HIF-1a) stimulates mucosal epithelium to increased production of mucins in dextran sodium sulfate (DSS) colitis. Colonic mRNA expression of (a) MUC1, (b) MUC2, and (c) MUC3 was significantly higher in DSS-treated CD11cCre/HIF-1a þ f/ þ f mice than in HIF-1a þ f/ þ f DSS-treated mice or in mice not treated with DSS. (d) Sections (4 mm) of colon tissue were stained with MUC2 (red) and 4’,6-diamidino-2-phenylindole (DAPI; blue). After treatment with DSS, the amount of colonic MUC2 protein was higher in CD11cCre/HIF- 1a þ f/ þ f mice than in HIF-1a þ f/ þ f mice (original magnification 200; scale bar ¼ 50 mm). N ¼ 5–7 per group. *Po0.05; **Po0.01; ****Po0.0001. C, control; NS, not significant.

Article Snippet: MUC2 was detected with a rabbit anti-mouse MUC2 antibody (Santa Cruz Biotechnology).

Techniques: Knock-Out, Expressing, Staining, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sialic acid plays a pivotal role in licensing Citrobacter rodentium's transition from the intestinal lumen to a mucosal adherent niche.

doi: 10.1073/pnas.2301115120

Figure Lengend Snippet: Fig. 1. Citrobacter rodentium resides within the colonic mucus and catabolizes sialic acid. (A) C. rodentium localizes to the mucus layer. Representative immunofluorescence staining of mouse colonic tissue infected with C. rodentium. A colon cross-section (green panel) was stained with DAPI to detect DNA (blue), anti-C. rodentium (red) to visualize C. rodentium, and anti-Muc2 to visualize mucus (green). The gray panel is the enlarged view of the boxed region within the cross-section, original magnification = 200×. The orange panel is a magnified image indicating a subpopulation of C. rodentium localized to the inner mucus and traversing the mucus (arrowheads), with a separate image showing C. rodentium staining independently (red channel), original magnification = 630×. (Scale bar, 15 μm.) (B) C. rodentium uses sialic acid as a sole carbon source for growth. C. rodentium growth was measured by optical density (OD600) at 20-min intervals over 24 h at 37 °C in M9 minimal medium supplemented with 0.2% N-acetylneuraminic acid (sialic acid) or purified mucins. Data are presented as averages of cell growth (n = 9) from three independent experiments.

Article Snippet: For visualizing C. rodentium localization in the mucus, methacarn- fixed mouse distal colons were stained with the following primary antibodies—rat anti- C. rodentium Tir (gift from W. Deng), rabbit anti- Muc2 (Boster), and rabbit anti- Muc2 (Novus), which were then probed with Alexa Fluor 488- conjugated donkey anti- rabbit IgG (Life Technologies) and Alexa Fluor 568–conjugated donkey anti- rat IgG (Life Technologies).

Techniques: Immunofluorescence, Staining, Infection, Purification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sialic acid plays a pivotal role in licensing Citrobacter rodentium's transition from the intestinal lumen to a mucosal adherent niche.

doi: 10.1073/pnas.2301115120

Figure Lengend Snippet: Fig. 2. Sialic acid in the colon is mainly derived from mucus produced by goblet cells and widely expressed before and during C. rodentium infection. (A) Representative immunofluorescence staining of sialic acid on murine colonic sections with and without C. rodentium infection. Sections were stained with DAPI to detect DNA (blue) and SNA lectin (α2,6-sialic acid binding) to visualize sialic acid. Dotted lines indicate the apical side of the epithelium. Original magnification = 200×. (Scale bar, 50 µm.) (B) Degree of sialylation on Muc2 O-glycans of colonic mucus with (n = 4) and without (n = 4) infection with C. rodentium for 6 d. Released O-glycans from distal intestine were analyzed on PGC-LC-MS/MS. (C) Levels of free sialic acid in fecal contents of mice without (n = 6) and with (n = 6) C. rodentium infection for 6 d. All data are shown as mean ± SEM. Statistical significance calculated by the Mann–Whitney U test (B and C).

Article Snippet: For visualizing C. rodentium localization in the mucus, methacarn- fixed mouse distal colons were stained with the following primary antibodies—rat anti- C. rodentium Tir (gift from W. Deng), rabbit anti- Muc2 (Boster), and rabbit anti- Muc2 (Novus), which were then probed with Alexa Fluor 488- conjugated donkey anti- rabbit IgG (Life Technologies) and Alexa Fluor 568–conjugated donkey anti- rat IgG (Life Technologies).

Techniques: Derivative Assay, Produced, Infection, Immunofluorescence, Staining, Binding Assay, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY

Journal: Gut Microbes

Article Title: Defining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus

doi: 10.1080/19490976.2025.2494717

Figure Lengend Snippet: The ALI model produces a physiologically relevant mucus layer. a, The experimental procedure to generate ALI-derived mucus, created with Biorender.com. b, The formation of the mucus layer after growing ALI cultures for 7 days, 14 days and 21 days. Arrows indicate the mucus layer. The average mucus thickness at 14 days and 21 days is 114 ± 28 µm, and 140 ± 25 µm, respectively. c, Immunostaining of Muc2 -/- ALI (at 21 days). ALI cross-sections were stained with DAPI (blue), anti-Muc2 (green) and anti-E-cadherin (white). d, Proteomics analysis of gel-forming mucins present in mouse ALI-derived mucus. e-f, Pie chart of acidic (e) and various subsets (f) of mucin-type O-glycans in colonic ALI-derived mucus. g, MALII (red) staining of Muc2 +/+ ALI culture. Scale bar, 200 µm.

Article Snippet: Muc2 degradation products were detected by immunoblotting using an anti-Muc2 rabbit polyclonal antibody (Novus Biologicals, NBP1–31231) (1:1,000) which targets an epitope on the C terminus of mucin 2 from both human and mouse origin.

Techniques: Derivative Assay, Immunostaining, Staining

Journal: Gut Microbes

Article Title: Defining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus

doi: 10.1080/19490976.2025.2494717

Figure Lengend Snippet: The ALI model is able to recapitulate pathogen-mucus interaction in vivo . a, C. rodentium infection of Muc2 +/+ ALI cultures for 6 h and 10 h. Scale bar, 200 µm. b, C. rodentium infection of Muc2 -/- ALI culture for 6 h. ALI cross-sections were stained with anti- C. rodentium LPS (red), Ulex europaeus agglutinin-1 (UEA-1, green), DAPI (blue) and E-cadherin (white). Scale bar, 200 µm. White arrows indicate C. rodentium present in the mucus. Yellow arrows indicate C. rodentium close to IECs. Yellow arrowheads indicate cell sloughing.

Article Snippet: Muc2 degradation products were detected by immunoblotting using an anti-Muc2 rabbit polyclonal antibody (Novus Biologicals, NBP1–31231) (1:1,000) which targets an epitope on the C terminus of mucin 2 from both human and mouse origin.

Techniques: In Vivo, Infection, Staining

Journal: Gut Microbes

Article Title: Defining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus

doi: 10.1080/19490976.2025.2494717

Figure Lengend Snippet: Sialic acid enhances C. rodentium ’s ability to degrade ALI-derived mucus and infect IECs. a, Diagram of mucus degradation assay in vitro , created with Biorender.com. b, Mucus degradation assay using the supernatant from C. rodentium grown in media containing 0.45% glucose (Glc) or sialic acid (SA). Mucus incubated with C. rodentium supernatants was loaded onto 3–8% Tris-acetate gels and run through electrophoresis. Proteins were visualized by western blot using an anti-Muc2 antibody. c, Quantification analysis of degraded Muc2 band to total Muc2 band, ***, p < 0.001; ****, p < 0.0001. d, Immunostaining of ALI cultures infected with C. rodentium in the presence of glucose or sialic acid. ALI cross-sections were stained with anti- C. rodentium LPS (red), UEA-1(green), DAPI (blue) and E-cadherin (white). Yellow arrows indicate C. rodentium close to IECs. Yellow arrowheads indicate cell sloughing. Scale bar, 200 µm.

Article Snippet: Muc2 degradation products were detected by immunoblotting using an anti-Muc2 rabbit polyclonal antibody (Novus Biologicals, NBP1–31231) (1:1,000) which targets an epitope on the C terminus of mucin 2 from both human and mouse origin.

Techniques: Derivative Assay, Degradation Assay, In Vitro, Incubation, Electrophoresis, Western Blot, Immunostaining, Infection, Staining

Journal: Gut Microbes

Article Title: Defining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus

doi: 10.1080/19490976.2025.2494717

Figure Lengend Snippet: A comparison of the ability of WT, Δpic, ΔespC and ΔpicΔespC C. rodentium strains to infect mouse ALI cultures and to degrade the ALI-derived mucus. a, The ALI cultures were infected with different C. rodentium strains, including WT, Δpic, ΔespC and ΔpicΔespC double mutant ( ΔΔ ). ALI cross-sections were stained with anti- C. rodentium LPS (red), UEA-1(green), DAPI (blue) and E-cadherin (white). White arrows indicate C. rodentium on the top of the mucus. Scale bar, 200 µm. b, Diagram of mucus degradation assay after C. rodentium infection, created with Biorender.com. ALI cultures were infected with different bacterial strains, followed by the collection of mucus from infected cultures. c, mucus from infected ALI was collected and run on a 3%-8% Tris-acetate gel, followed by western blot using an anti-Muc2 antibody.

Article Snippet: Muc2 degradation products were detected by immunoblotting using an anti-Muc2 rabbit polyclonal antibody (Novus Biologicals, NBP1–31231) (1:1,000) which targets an epitope on the C terminus of mucin 2 from both human and mouse origin.

Techniques: Comparison, Derivative Assay, Infection, Mutagenesis, Staining, Degradation Assay, Western Blot

Journal: Gut Microbes

Article Title: Defining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus

doi: 10.1080/19490976.2025.2494717

Figure Lengend Snippet: Mucus degradation assay using protein secreted by SPATEs expressing E. coli DH5α. a, The degradation of mouse ALI-derived mucus by supernatant from E. coli DH5α expressing CrespC or Crpic . EV, empty vector; L, protein ladder; E, empty lane. Note, the anti-Muc2 antibody shows cross-activity against Pic (~110 kDa). *, p < 0.05. b, The degradation of mouse ALI-derived mucus by supernatant from E. coli DH5α expressing CrespC or CrespC-S251I . c, The degradation of mouse ALI-derived mucus by supernatant from E. coli DH5α expressing CrespC or EPEC espC . Samples from the mucus degradation assay were reduced and run on a 3%-8% Tris-acetate gel, followed by western blot using an anti-C-terminus Muc2 antibody.

Article Snippet: Muc2 degradation products were detected by immunoblotting using an anti-Muc2 rabbit polyclonal antibody (Novus Biologicals, NBP1–31231) (1:1,000) which targets an epitope on the C terminus of mucin 2 from both human and mouse origin.

Techniques: Degradation Assay, Expressing, Derivative Assay, Plasmid Preparation, Activity Assay, Western Blot

Journal: Gut Microbes

Article Title: Defining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus

doi: 10.1080/19490976.2025.2494717

Figure Lengend Snippet: Recombinant CrEspC is able to degrade mouse ALI-derived mucus. a, The sequence information used to express recombinant CrEspC by E. coli BL21 Star (DE3) system, created with Biorender.com. SS, signal sequence. b, The degradation of mouse ALI-derived mucus by 0.05 µg purified CrEspC for 2 h, 8 h and 24 h. c, The degradation of mouse ALI-derived mucus by different amounts (0.0025, 0.0125, 0.05 µg) of purified CrEspC for 2 h. Samples were reduced and run on a 3%-8% Tris-acetate gel, followed by western blot using an anti-C-terminus Muc2 antibody.

Article Snippet: Muc2 degradation products were detected by immunoblotting using an anti-Muc2 rabbit polyclonal antibody (Novus Biologicals, NBP1–31231) (1:1,000) which targets an epitope on the C terminus of mucin 2 from both human and mouse origin.

Techniques: Recombinant, Derivative Assay, Sequencing, Purification, Western Blot

Journal: Journal of Animal Science and Technology

Article Title: Functional characteristics of a pig 2D intestinal organoid model as an in vitro platform for nutritional studies

doi: 10.5187/jast.2024.e116

Figure Lengend Snippet: (A) Expression of the mRNA levels of epithelial cell marker genes in IPEC-J2 and pig intestinal organoids. Data are presented as mean ± SD (n = 3–5). (B) Immunostaining of MUC2 in IPEC-J2 and pig intestinal organoids. (C) Immunostaining of CHGA in IPEC-J2 and pig intestinal organoids. Nucleus and F-actin were stained with Hoechst 33342 and phalloidin. Scale bar = 50 μm. * p < 0.05. ND, non-detected.

Article Snippet: The primary antibodies rabbit anti-Muc2 (27675-1-AP, ProteinTech) and mouse anti-ChgA (sc-393941, Santa Cruz Biotechnology) were diluted at 1:50 and 1:100, respectively, with the blocking buffer.

Techniques: Expressing, Marker, Immunostaining, Staining