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: Nutrients

Article Title: Xenogeneic-Free Human Intestinal Organoids for Assessing Intestinal Nutrient Absorption

doi: 10.3390/nu14030438

Figure Lengend Snippet: The structures and properties of XF-HIOs. ( A ) A photograph of three-dimensional xenogeneic-free human intestinal organoids (XF-HIOs). ( B ) The level of expression of mRNAs for LGR5 , Villin , and CDX2 relative to that of the human small intestine. Data shown represent the mean ± S.E. values of three independent experiments. n.s.; not significant. ( C ) Immunofluorescent images of XF-HIOs with anti-VILLIN, CDX2, and MUCIN2 (MUC2). DAPI, 4′, 6-diamidino-2-phenylindole. Scale bars: 50 μm.

Article Snippet: Secreted Muc2 was assessed using Human Muc2 ELISA Kit (Elabscience, TX, USA).

Techniques: Expressing

Journal: Immunology

Article Title: Human Gut Commensal Bacteroides fragilis Suppresses Mucin Production and Alters Microbiota Composition Resulting in Accelerated Type 1 Diabetes in Mice

doi: 10.1111/imm.70032

Figure Lengend Snippet: Suppressed mucin production in the distal colon of conventionalized BF‐colonised mice. Sections of formalin fixed distal colon and ileum tissues (with luminal contents) of 12‐week‐old conventionalized GF (GF‐conv/Ex‐GF) and conventionalized BF‐monocolonised (BF‐conv/Ex‐BF) mice were subjected to immunofluorescence staining using anti‐Muc2 antibody, or Alcian blue/nuclear fast, periodic acid‐Schiff's/haematoxylin, and Alcian blue periodic acid‐Schiff's/haematoxylin staining. (A) Representative images of immunofluorescence staining of colon and ileum tissue sections (left panels) and relative fluorescence values of Muc2 staining of multiple sections of distal colon tissues from 3 mice/group (right panel) are shown. (B) Representative images Alcian blue/nuclear fast, periodic acid‐Schiff's/haematoxylin, and Alcian blue periodic acid‐Schiff's/haematoxylin staining of colon and ileum tissue sections are shown.

Article Snippet: Tissue sections were stained using a mouse Muc2 specific antibody (Novus Biologicals) followed by an Alexaflor‐488 linked secondary antibody.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Redox biology

Article Title: Glutamine promotes O-GlcNAcylation of G6PD and inhibits AGR2 S-glutathionylation to maintain the intestinal mucus barrier in burned septic mice.

doi: 10.1016/j.redox.2022.102581

Figure Lengend Snippet: Fig. 2. Gln supplementation promoted MUC2 maturation and attenuated colonic mucus barrier damage after burn injury (A, B, C, D) Exploration of the expression of mature MUC2 in distal colon tissue at 3, 5, and 7 days post-injury from the sham, BI and BI + Gln groups. (E, F, G, H) Quantification of the expression of mature MUC2 in distal colon tissue at 3, 5, and 7 days post-injury. (N = 5 per group). (I, K) The expression of mature MUC2 was explored and quantified in HT-29 CL.16E cells with or without 2 mM Gln for 12 h (N = 6 per group). (J, L) The expression of mature MUC2 was explored and quantified in HT-29 CL.16E cells. The cells were exposed for 12 h to LPS (100 ng/ml), and cultured in the absence or presence of 2 mM Gln (N = 6 per group). (M) Immunostaining of colon sections using antibodies against MUC2-C (green) and FISH (with bacterial 16S rRNA gene probe) at 5 days post-injury. The red arrow represents bacterial staining. The inner stratified mucus layer (s) and outer mucus layer (o) are marked with a white segmented line. Blue indicates nuclear (DAPI) staining (scale bar: 50 μm). . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The cells were cultured on small round cover glasses, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with goat serum, and treated with antibodies against MUC2-VNTR (Novus, NBP2-25221), MUC2-N (Abcam, ab90007), AGR2 (Abcam, ab76473) and glutathione (Virogen, 101-A) overnight at 4 ◦C.

Techniques: Expressing, Cell Culture, Immunostaining, Staining

Journal: Redox biology

Article Title: Glutamine promotes O-GlcNAcylation of G6PD and inhibits AGR2 S-glutathionylation to maintain the intestinal mucus barrier in burned septic mice.

doi: 10.1016/j.redox.2022.102581

Figure Lengend Snippet: Fig. 3. S-glutathionylation of AGR2 restricted the processing of MUC2. (A) AGR2 was immunoprecipitated from HT-29 CL.16E cells, and associated MUC2 was detected by immunoblotting. Rabbit or mouse IgG was used as a control. A total of 5% of the volume of lysate used for the immunoprecipitation was run on the gel for comparison (input). (B) Immunofluorescent localization of the MUC2 precursor (green) and AGR2 (red) in HT-29 CL.16E cells. Blue indicates nuclear (DAPI) staining. Scale bar: 10 μm. (C) The expression levels of the MUC2 precursor, mature MUC2 and AGR2 were assessed in AGR2 Knockout HT-29 CL.16E cell line. (D) Immunofluorescence staining for the MUC2 precursor (green) and mature MUC2(red) in AGR2 Knockout HT-29 CL.16E cell line. Blue indicates nuclear (DAPI) staining. Scale bar: 10 μm. (E) Expression of mature MUC2, immature MUC2, AGR2, GRP78, and CHOP in the distal colons of sham, burn–infected mice at 5 days post-injury (N = 5 per group). (F) Staining for ER-localized MUC2 precursor (green) in mouse goblet cells to assess MUC2 biosynthesis at 5 days post-injury. Scale bar: 50 μm. (G) Representative confocal microscopy images of distal colon sections for analysis of AGR2 (red) and MUC2 (green) at 5 days post-injury, with yellow arrows representing immature MUC2 and red arrows indicating mature MUC2. Scale bar: 50 μm. (H) Cysteine thiol levels of AGR2 in the distal colons of sham and burn-infected mice at 5 days post-injury. Tissue lysates were prepared, incubated with Protein-SHifter Plus, and subjected to SDS‒PAGE without BME (N = 3 per group). (I) Immunoblotting for S-glutathionylation of protein in colon tissues (DTT, negative control; GAPDH, loading control; N = 4 per group). (J) Co-IP showing S-glutathionylated AGR2 in colon tissue lysates from sham or burn- infected mice at 5 days post-injury (IP, GSH; IB, AGR2). (K) Tissue lysates were incubated with a MUC2-VTNR antibody coupled to Thermo Scientific Pierce Pro tein A/G magnetic beads for 24 h. The precipitates were subjected to Western blotting with anti-MUC2-VTNR and anti-AGR2 antibodies. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The cells were cultured on small round cover glasses, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with goat serum, and treated with antibodies against MUC2-VNTR (Novus, NBP2-25221), MUC2-N (Abcam, ab90007), AGR2 (Abcam, ab76473) and glutathione (Virogen, 101-A) overnight at 4 ◦C.

Techniques: Immunoprecipitation, Western Blot, Control, Comparison, Staining, Expressing, Knock-Out, Immunofluorescence, Infection, Confocal Microscopy, Incubation, Negative Control, Co-Immunoprecipitation Assay, Magnetic Beads

Journal: Redox biology

Article Title: Glutamine promotes O-GlcNAcylation of G6PD and inhibits AGR2 S-glutathionylation to maintain the intestinal mucus barrier in burned septic mice.

doi: 10.1016/j.redox.2022.102581

Figure Lengend Snippet: Fig. 4. Gln mitigated LPS-induced oxidative stress and S-glutathionylation of AGR2, which regulates MUC2 processing. HT-29 CL.16E cells were cultured in the absence or presence of Gln (2 mM) and LPS (100 ng/ml) for 8 h. (A) The fluorescence intensity in HT-29 CL.16E cells was measured by flow cytometry using H2DCFDA dye (N = 6 per group). (B) The intracytoplasmic superoxide anion concentration in HT-29 CL.16E cells was assessed by flow cytometry using a dihydroethidium (DHE) probe (N = 6 per group). (C) Quantitative estimation of LPS-induced H2O2. Hydrogen peroxide released by cells was detected with the ROS-Glo™ Hydrogen Peroxide Kit, which was calculated by comparing the hydrogen peroxide standard curve. (D) GSH levels in cell lysates (N = 5 per group). (E) GSH/GSSG ratio in cell lysates (N = 5 per group). (F) Immunoblotting analysis of the cysteine thiol levels of AGR2 under different cellular treatments. HT-29 CL.16E cells were exposed for 30 min to H2O2 (200 μM), then stimulated with or without 5 mM sodium pyruvate (SP), (DTT, positive control). (G) Nonreducing electrophoresis and Western blot analysis with streptavidin-HRP to determine the S-glutathionylation level of the protein. HT-29 CL.16E cells were preloaded with BioGEE (250 μM) for 1.5 h and stimulated with H2O2 (200 μM) for 30 min, then cultured in the presence and absence of 5 mM sodium pyruvate (DTT, negative control). (H) Reduction electrophoresis and Western blot to determine the S-glutathionylation level of AGR2 after pulling down the biotin-GSS-protein adducts. (I) Immunoblots of AGR2 glutathionylation under different cellular treatments. HT-29 CL.16E cells were preloaded with BioGEE (250 μM) for 1.5 h, stimulated with H2O2 (200 μM) for 30 min, and then incubated with or without 2 mM N-acetylcysteine (DTT, negative control). (J) Western blot analysis with streptavidin-HRP to determine the S-glutathionylation level of protein. HT-29 CL.16E cells were preloaded with BioGEE (250 μM) for 1.5 h and exposed for 8 h to LPS (100 ng/ml), next cultured in the absence or presence of Gln (2 mM) and 2 mM N-acetylcysteine (DTT, negative control). (K) Immunoblotting to determine the S- glutathionylation level of AGR2 after pulling down the biotin-GSS-protein adducts. (L) Immunoblotting analysis of the cysteine thiol levels of AGR2 under different cellular treatments. HT-29 CL.16E cells were exposed for 8 h to LPS (100 ng/ml), and cultured in the absence or presence of 2 mM Gln (DTT, positive control; H2O2, negative control; N = 3 per group). (M) Confocal imaging of glutathionylated proteins. HT-29 CL.16E cells were cultured in the absence or presence of LPS and Gln 8 h. Green fluorescent labeling was performed using an anti-glutathionylated antibody. Blue indicates nuclear (DAPI) staining. Scale bar, 10 μm. (N) The expression of mature MUC2, immature MUC2, GRP78 and CHOP was analysed by immunoblotting. HT-29 CL.16E cells were cultured in the absence or presence of LPS and Gln (N = 3 per group). * Compared with control group, # Compared with LPS group, #P < 0.05; # #P < 0.01; # # #P < 0.001. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The cells were cultured on small round cover glasses, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with goat serum, and treated with antibodies against MUC2-VNTR (Novus, NBP2-25221), MUC2-N (Abcam, ab90007), AGR2 (Abcam, ab76473) and glutathione (Virogen, 101-A) overnight at 4 ◦C.

Techniques: Cell Culture, Fluorescence, Flow Cytometry, Concentration Assay, Western Blot, Positive Control, Electrophoresis, Negative Control, Incubation, Imaging, Labeling, Staining, Expressing, Control

Journal: Redox biology

Article Title: Glutamine promotes O-GlcNAcylation of G6PD and inhibits AGR2 S-glutathionylation to maintain the intestinal mucus barrier in burned septic mice.

doi: 10.1016/j.redox.2022.102581

Figure Lengend Snippet: Fig. 6. O-GlcNAcylation of G6PD regulates its activity through dimerization to promote MUC2 maturation. (A) Immunoblot analysis of protein O-GlcNAc modification in the distal colons of sham and burn-infected mice at 5 days post-injury (N = 5 per group). (B) Detection of O-GlcNAcylation of G6PD in the distal colons of sham and burn-infected mice by using chemical enzyme labeling and biotinylation (N = 5 per group). (C) G6PD enzyme activity assay (N = 5 per group). (D) Labeling of G6PD glycosylation levels in WT and S84V G6PD cells. (E) Immunoblot for O-GlcNAcylation of G6PD. Cells labelled WT and S84V were treated with DON (20 μM) and TMG (50 μM). (F) G6PD enzyme activity assay of cells under different treatments (N = 5 per group). (G) Oligomerization status in WT and S84V G6PD cells measured by immunoblotting. Cross-linking with 1 mM DSS. (H) Immunoblot for O-GlcNAcylation of G6PD. WT and S84V G6PD cells were exposed to LPS (100 ng/mL) for 6 h and treated with or without NADPH. (I) G6PD enzyme activity assay under different treatments (N = 5 per group). (J) GSH levels in cell lysates (N = 5 per group). (K) GSH/GSSG ratio in cell lysates (N = 5 per group). (L) Co-IP showing S-glutathionylated AGR2 in cell lysates (N = 3 per group). (M) Immunoblot detection of mature MUC2 and immature MUC2 (N = 3 per group). * Compared with the WT group, # Compared with the S84V group without NADPH, #P < 0.05; # #P < 0.01; # # #P < 0.001.

Article Snippet: The cells were cultured on small round cover glasses, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with goat serum, and treated with antibodies against MUC2-VNTR (Novus, NBP2-25221), MUC2-N (Abcam, ab90007), AGR2 (Abcam, ab76473) and glutathione (Virogen, 101-A) overnight at 4 ◦C.

Techniques: Activity Assay, Western Blot, Modification, Infection, Labeling, Enzyme Activity Assay, Glycoproteomics, Co-Immunoprecipitation Assay

Journal: Redox biology

Article Title: Glutamine promotes O-GlcNAcylation of G6PD and inhibits AGR2 S-glutathionylation to maintain the intestinal mucus barrier in burned septic mice.

doi: 10.1016/j.redox.2022.102581

Figure Lengend Snippet: Fig. 7. Gln activated G6PD activity and stabilized AGR2 redox homeostasis via the HBP (A) Hexosamine biosynthetic pathway. (B, C) Effects of Gln and glucosamine on G6PD protein and O-GlcNAcylation levels as determined by immunoblotting of G6PD protein with antibodies against the G6PD protein and O-GlcNAc. HT-29 CL.16E cells were treated with 2 mM Gln or 10 mM glucosamine for 12 h, and cultured in the absence or presence of DON (20 μM). (D) Immunoblot detection of cellular protein O-GlcNAcylation. HT-29 CL.16E cells were exposed for 12 h to LPS (100 ng/ml), cultured in the absence or presence of Gln (2 mM), and then stimulated with or without Don (N = 3 per group). (E) Chemical enzyme labeling and biotinylation to detect the O-GlcNAcylation of G6PD (N = 3 per group). (F) Immunoblot detection of G6PD dimers and monomers. Cells were cross-linked with 1 mM DSS (N = 3 per group). (G) A -SulfoBiotics- Protein Redox State Monitoring Kit was used to determine the thiol redox state of recombinant proteins (N = 3 per group). (H) S- glutathionylated AGR2/AGR2 was measured by immunoblot analysis (N = 3 per group).(I) Immunoblot detection of mature MUC2 and immature MUC2 protein expression (N = 3 per group). * Compared with control, & Compared with the LPS group, & P < 0.05; &&P < 0.01; &&& P < 0.001. # Compared with LPS + Gln group, #P < 0.05; # #P < 0.01; # # #P < 0.001.

Article Snippet: The cells were cultured on small round cover glasses, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with goat serum, and treated with antibodies against MUC2-VNTR (Novus, NBP2-25221), MUC2-N (Abcam, ab90007), AGR2 (Abcam, ab76473) and glutathione (Virogen, 101-A) overnight at 4 ◦C.

Techniques: Activity Assay, Western Blot, Cell Culture, Labeling, Recombinant, Expressing, Control

Journal: Redox biology

Article Title: Glutamine promotes O-GlcNAcylation of G6PD and inhibits AGR2 S-glutathionylation to maintain the intestinal mucus barrier in burned septic mice.

doi: 10.1016/j.redox.2022.102581

Figure Lengend Snippet: Fig. 8. Gln promoted MUC2 maturation by stabilizing AGR2 redox homeostasis induced by O-GlcNAcylation of G6PD in burn infection. (A) O-GlcNAcylation levels in the distal colons after 5 days of glutamine supplementation in burn-infected mice (N = 3 per group). (B) Chemical enzyme labeling and biotinylation to detect G6PD O-GlcNAcylation (N = 3 per group). (C) GSH levels in distal colon tissue lysates (N = 3 per group). (D) GSH/GSSG in distal colon tissue lysates (N = 3 per group). (E) Immunoblot analysis of the cysteine thiol level of AGR2. Tissue lysates were incubated with Protein-Shifter Plus and subjected to SDS‒PAGE in the absence of BME (N = 3 per group). (F) Immunoblot analysis of the S-glutathionylation levels of total protein (N = 3 per group). (G) Co-IP showing S-glutathionylated AGR2 in tissue lysates (N = 3 per group). * Compared with sham group, # Compared with BI group, #P < 0.05; # #P < 0.01; # # #P < 0.001. (H) Immunoblotting for the expression of mature MUC2, immature MUC2, GRP78 and CHOP in the distal colon (N = 5 per group). (I) Representative AGR2 and MUC2 protein staining in colon sections. The yellow arrows indicate immature MUC2, and the red arrows indicate mature MUC2. (J) Proposed mechanism for the effect of Gln on promoting MUC2 maturation in burn-infected mice. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The cells were cultured on small round cover glasses, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with goat serum, and treated with antibodies against MUC2-VNTR (Novus, NBP2-25221), MUC2-N (Abcam, ab90007), AGR2 (Abcam, ab76473) and glutathione (Virogen, 101-A) overnight at 4 ◦C.

Techniques: Infection, Labeling, Western Blot, Incubation, Co-Immunoprecipitation Assay, Expressing, Staining

Journal: Molecular medicine reports

Article Title: Rebamipide upregulates mucin secretion of intestinal goblet cells via Akt phosphorylation.

doi: 10.3892/mmr.2017.7647

Figure Lengend Snippet: Figure 2. Reb significantly increased MUC2 mRNA expression. Reb (10 µM) was added to LS174T, and the mRNA expressions of MUC2 were assessed by real time PCR. The graph shows mRNA level of MUC2/GAPDH 6 h (A) and 24 h (B) after Reb addition. Data were presented in mean ± SE out of the three experiments. #P<0.05. Reb, rebamipide; MUC2, mucin 2, oligomeric mucus/gel‑forming; PCR, polymerase chain reaction; NS, not significant.

Article Snippet: The blotted membrane was incubated with 1% bovine serum albumin (BSA)-Tris-buffered saline and incubated with anti-MUC2 antibody (1:3,000; Novus) overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Rebamipide upregulates mucin secretion of intestinal goblet cells via Akt phosphorylation.

doi: 10.3892/mmr.2017.7647

Figure Lengend Snippet: Figure 3. Reb significantly increased MUC2 secretion. Reb at various concentrations (1‑100 µM) was added to LS174T and MUC2 secretion in the culture supernatant was assessed by using dot blot method. (A) A representa tive image of the MUC2 bands was presented and (B) the graph of MUC2 densitometry data is shown. The mean density was quantified by Image J software. Data were presented as mean ± SE out of the three experiments. #P<0.05. Reb, rebamipide; MUC2, mucin 2, oligomeric mucus/gel‑forming.

Article Snippet: The blotted membrane was incubated with 1% bovine serum albumin (BSA)-Tris-buffered saline and incubated with anti-MUC2 antibody (1:3,000; Novus) overnight.

Techniques: Dot Blot, Software

Journal: Molecular medicine reports

Article Title: Rebamipide upregulates mucin secretion of intestinal goblet cells via Akt phosphorylation.

doi: 10.3892/mmr.2017.7647

Figure Lengend Snippet: Figure 7. Reb significantly increased MUC2 secretion via p‑Akt. Reb (10 µM) was added to LS174T, and MUC2 secretion at 24 h was assessed by the dot blot method in the presence or absence of two inhibitors; EGFR kinase inhibitor (AG1478, 200 nM) and PI3 kinase/Akt inhibitor (wort mannin, 10 µM). Inhibitors were added to LS174T cells 30 min prior to Reb addition. A representative image out of the three experiments was presented. The band density was presented in the lower panel. Data were presented as mean ± SE out of the three experiments. *P<0.05 vs. the control; #P<0.05 vs. Reb. Reb, rebamipide; MUC2, mucin 2, oligomeric mucus/gel‑forming; p‑Akt, phosphorylated Akt; EGFR, epidermal growth factor receptor.

Article Snippet: The blotted membrane was incubated with 1% bovine serum albumin (BSA)-Tris-buffered saline and incubated with anti-MUC2 antibody (1:3,000; Novus) overnight.

Techniques: Dot Blot, Control

Journal: Advanced functional materials

Article Title: A Pumpless, High-Throughput Microphysiological System to Mimic Enteric Innervation of Duodenal Epithelium and the Impact on Barrier Function

doi: 10.1002/adfm.202409718

Figure Lengend Snippet: Our MPS system enables traditional barrier strength measures like transepithelial electrical resistance (TEER) and apparent permeability assay. a) TEER was measured via chopstick electrodes and the EVOM2. b) TEER values were overall greater for cultures containing the epithelium (Epi); n = 3, m = 3; error bars = SD; ** = p < 0.01, one-way ANOVA with multiple comparisons. c) Fold change of the TEER values controlled for the 3D gel layer degrading when ENS was cultured in it; n = 3, m = 3; error bars = SD; * = p < 0.05; two-tailed unpaired t -test. d) Apparent permeability, measured by lucifer yellow diffusion through the culture, followed the TEER results. e) Less fluorophore traveled through the layers when there was an epithelium present (n = 3, m = 3; error bars = SD; ** = p < 0.01; one-way ANOVA with multiple comparisons) and f) the fold change comparison to appropriate controls showed a significant difference between the epithelium with and without the ENS (n = 3, m = 3; error bars = SD; * = p < 0.05, *** = p < 0.001; two-tailed unpaired t -test). g) Cell area coverage was also measured using phalloidin-stained monolayers and images of the entire culture area. h) The concentration of Muc2 is higher in the epithelium containing cultures, as well as the co-culture show a trending increase in Muc2 production compared to the epithelium alone (n = 3, m = 2–3; error bars = SD; * = p < 0.05; one-way ANOVA with multiple comparisons). i) A higher EGF basal supernatant concentration was recorded from the co-culture condition than either of the monoculture conditions (n = 3, m = 2–3; error bars = SD; * = p < 0.05; *** = p < 0.001; Kruskal–Wallis test for non-parametric data).

Article Snippet: A Muc2 ELISA ( Novus Biologicals , NBP276701) was performed similarly using the apical supernatants.

Techniques: Permeability, Cell Culture, Two Tailed Test, Diffusion-based Assay, Comparison, Staining, Concentration Assay, Co-Culture Assay