Journal: Scientific Reports

Article Title: Dietary polyamines promote intestinal adaptation in an experimental model of short bowel syndrome

doi: 10.1038/s41598-024-55258-4

Figure Lengend Snippet: Polyamines enhance mucosal defense factors in rats with massive intestinal resection. (A-B) Fecal and serum secretory IgA was measured using ELISA. n = 5–7/group. (C) IgA in ileum tissue was assessed using western blotting. n = 4–6/group. (D) Fecal mucin was measured using a fluorometric assay. n = 4–6/group. (E) Representative images of ileal villus showing immunostaining by Muc2 (original magnification, ×400; scale bars = 200 μm). Left graphs show the number of goblet cells per unit villous area and the size of goblet cell secretion granule. (F) The expression of Claudin-3 in the ileum tissue was measured by western blot analysis. Representative images of ileal villus showing immunostaining by Claudin-3 (original magnification, ×200; scale bars = 100 μm). (G) Serum DAO was measured by ELISA. n = 5–7/group. (H) Serum GLP-2 was measured by ELISA. n = 5–7/group. (I) Fecal short-chain fatty acid (SCFA) content was measured using high-performance liquid chromatography. n = 3–6/group. Data are presented as mean ± SD. Results of one-way ANOVA are represented as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Nonspecific antibody binding was blocked using 15% goat serum for 30 min. For staining of Ki-67, Muc2, and Claudin-3, the slides were then incubated with anti-Ki67 antibody (ab16667; Abcam, Cambridge, UK), anti-Muc2 rabbit monoclonal antibody (M01212-1; Boster Biological Technology, Pleasanton, CA, USA), and Claudin-3 polyclonal antibody (#PA5-32353; Invitrogen, Waltham, MA, USA) at 4°C overnight, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunostaining, Expressing, High Performance Liquid Chromatography

Journal: Investigative Ophthalmology & Visual Science

Article Title: Ectoine Enhances Mucin Production Via Restoring IL-13/IFN-γ Balance in a Murine Dry Eye Model

doi: 10.1167/iovs.65.6.39

Figure Lengend Snippet: Antibodies Used in This Study

Article Snippet: MUC2 , CUSABIO , PA207869 , Rabbit , 1:3000.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Ectoine Enhances Mucin Production Via Restoring IL-13/IFN-γ Balance in a Murine Dry Eye Model

doi: 10.1167/iovs.65.6.39

Figure Lengend Snippet: Mouse TaqMan Gene Expression Assays Used in This Study

Article Snippet: MUC2 , CUSABIO , PA207869 , Rabbit , 1:3000.

Techniques: Gene Expression

Journal: Investigative Ophthalmology & Visual Science

Article Title: Ectoine Enhances Mucin Production Via Restoring IL-13/IFN-γ Balance in a Murine Dry Eye Model

doi: 10.1167/iovs.65.6.39

Figure Lengend Snippet: Ectoine enhances production of secreted mucins in DS mice. ( A , C ) Representative images of IF staining showing the decreased production of MUC5AC ( A ) and MUC2 ( C ) in DS mice (DS+PBS) was recovered by ectoine eye drops (DS+Ect); Scale bars = 50 µm. The stitched images were made because the cornea and conjunctiva in some eyeball sections were too far apart to be captured on the same image. ( B , D ) IF intensity quantified the alternation of MUC5AC and MUC2 in 3 groups; each circle represents one eye. Data are shown as Mean ± SD, n = 6; the P values are shown in the graph as compared with the DS+PBS group.

Article Snippet: MUC2 , CUSABIO , PA207869 , Rabbit , 1:3000.

Techniques: Staining, Eye Drops

Journal: Investigative Ophthalmology & Visual Science

Article Title: Ectoine Enhances Mucin Production Via Restoring IL-13/IFN-γ Balance in a Murine Dry Eye Model

doi: 10.1167/iovs.65.6.39

Figure Lengend Snippet: Gene expression of MUC5AC, MUC2, MUC1, MUC4, MUC16 , and MUC15 by conjunctiva and cornea. ( A ) Gene expression of all 6 mucins in normal mice were detected at different mRNA levels based on the threshold cycle (Ct) value in real-time PCR with house-keeping gene GAPDH as an internal control. ( B ) The mRNA levels of all six mucins in conjunctiva of the three groups of mice. ( C ) The mRNA expression of MUC1, MUC4, and MUC16 by corneas in the three groups of mice. Data are shown as Mean ± SD, n = 6; the P values are shown in the graph as compared with the DS+PBS group.

Article Snippet: MUC2 , CUSABIO , PA207869 , Rabbit , 1:3000.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Control, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Intestinal changes in permeability, tight junction and mucin synthesis in a mouse model of Alzheimer's disease

doi: 10.3892/ijmm.2023.5316

Figure Lengend Snippet: Monoclonal primary antibodies in western blotting.

Article Snippet: Sections were immunostained with a rabbit anti-MUC2 antibody (1:100; red; cat. no. #DF8390; Affinity Biosciences) and incubated at 4°C overnight.

Techniques: Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Intestinal changes in permeability, tight junction and mucin synthesis in a mouse model of Alzheimer's disease

doi: 10.3892/ijmm.2023.5316

Figure Lengend Snippet: Variation in number of GCs and MUC2 expression in intestinal mucosa. (Aa-h) Alcian blue-Periodic acid Schiff staining showed GCs and (Ba-h) immunohistochemistry showed MUC2 in the ileum and proximal colon of WT and dTg mice at 6 and 12 months. (C) Number of GCs, as determined by histochemical staining of the ileum and proximal colon. (D) MUC2 expression area in the ileum and proximal colon was determined by immunohistochemistry (n=6/group). Scale bar, 50 µ m. (E) Protein levels of MUC2 in extracts of intestinal tissue were detected by western blotting. (F) Quantification of the western blot results (n=4/group). * P<0.05, ** P<0.01. GC, goblet cell; WT, wild-type; dTg, double transgene; MUC2, mucin 2; WGA, wheat germ agglutin.

Article Snippet: Sections were immunostained with a rabbit anti-MUC2 antibody (1:100; red; cat. no. #DF8390; Affinity Biosciences) and incubated at 4°C overnight.

Techniques: Expressing, Staining, Immunohistochemistry, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Intestinal changes in permeability, tight junction and mucin synthesis in a mouse model of Alzheimer's disease

doi: 10.3892/ijmm.2023.5316

Figure Lengend Snippet: Variation in MUC2 and WGA in intestinal mucosa. (Aa-p) IF showed MUC2 and WGA in ileum of WT and dTg mice at 6 and 12 months. (B) MUC2- and (C) WGA-positive area was determined by IF in ileum (n=6/group). IF showed MUC2 and WGA in (Da-p) proximal colon of WT and dTg mice at 6 and 12 months. (E) MUC2- and (F) WGA-positive area was determined by IF in proximal colon (n=6/group). * P<0.05, ** P<0.01. WGA, wheat germ agglutinin; IF, immunofluorescence; MUC2, mucin 2; WT, wild-type; dTg, double transgene.

Article Snippet: Sections were immunostained with a rabbit anti-MUC2 antibody (1:100; red; cat. no. #DF8390; Affinity Biosciences) and incubated at 4°C overnight.

Techniques: Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes

doi: 10.1083/jcb.200311021

Figure Lengend Snippet: SOX9 negatively regulates the promoters of the CDX2 and MUC2 genes. LS174T cells were cotransfected, in triplicate, with the indicated expression constructs (10 and 100 ng) together with CDX2-luciferase, MUC2-luciferase and SOX-luciferase reporters (500 ng) and relative luciferase activities were measured 36 h after transfection. The reporter activity in mock-transfected cells was arbitrarily set to 100. (a) CDX2 and MUC2 promoters regulation by SOX9; (b–d) SOX9-VP16 recapitulates the transcription regulation properties of the wild-type SOX9 on SOX-luciferase (b), CDX2-luciferase (c), and MUC2-luciferase constructs (d). The histograms represent mean values of triplicate experiments and SDs are shown with error bars.

Article Snippet: The MUC2 antibody (1:1,000) was provided by I. van Seuningen (INSERM U560, Lille, France). β-Actin A5441 and Flag (1:500) were purchased from Sigma-Aldrich; Ki-67 was purchased from Novocastra (1:150); β-catenin was purchased from BD Transduction Laboratories (1:50); CDX2 was purchased from Biogenex (1:500); and Myc tag 9E10 was purchased from Santa Cruz Biotechnology, Inc. (1:100).

Techniques: Expressing, Construct, Luciferase, Transfection, Activity Assay

Journal: The Journal of Cell Biology

Article Title: SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes

doi: 10.1083/jcb.200311021

Figure Lengend Snippet: SOX9 represses CDX2 and MUC2 in xenograft-derived tumors. Sections from tumors resulting from xenografts of HT29-16E-SOX9 cells, stained with antibodies against the Flag tag, the CDX2 and MUC2 proteins. Antibodies used for staining are indicated. The expression of the Flag-SOX9 construct is detected uniquely in the nucleus of tumors from doxycycline-fed mice. Bar, 130 μm.

Article Snippet: The MUC2 antibody (1:1,000) was provided by I. van Seuningen (INSERM U560, Lille, France). β-Actin A5441 and Flag (1:500) were purchased from Sigma-Aldrich; Ki-67 was purchased from Novocastra (1:150); β-catenin was purchased from BD Transduction Laboratories (1:50); CDX2 was purchased from Biogenex (1:500); and Myc tag 9E10 was purchased from Santa Cruz Biotechnology, Inc. (1:100).

Techniques: Derivative Assay, Staining, FLAG-tag, Expressing, Construct

Journal: bioRxiv

Article Title: Phosphatase-independent suppression of mucosal inflammation and disease progression by Salmonella SopB

doi: 10.1101/2024.09.02.610793

Figure Lengend Snippet: (A) Small intestinal tissue sections stained with H&E of non-infected (n=3) neonates or neonates infected with wt (n=3) or Δ sopB (n=3) S . Typhimurium at day 2 p.i.. Bar=100 µm. (B) Quantification of the depth of the lamina propria as a measure of the tissue edema measured in 15-20 different areas of one tissue section per animal (non-infected neonates (n=3) or neonates infected with wt (n=3) or Δ sopB (n=3)). (C) Muc2 immunostaining (red) in small intestine tissue sections of non-infected (n=3) neonates and the neonates infected with wt (n=3) or Δ sopB (n=3) S . Typhimurium at day 2 p.i.. Counterstaining with WGA (white), DAPI (blue); autofluorescence (green). Bar=50 µm. (D) Percentage of goblet cells among intestinal epithelial cells in non-infected (n=3) neonates and neonates infected with wt (n=3) or Δ sopB (n=3) S . Typhimurium at day 2 p.i.. 10-20 images with the size of 312µm x 250µm were evaluated on one tissue section per animal. (E) Goblet cell size in tissue sections of uninfected (n=3) neonates and neonates infected with wt (n=3) or Δ sopB (n=3) S . Typhimurium at day 2 p.i.. 11-21 goblet cells (µm 2 ) were analyzed on each section. (F) TUNEL staining (red) of neonatal small intestine tissue sections of non-infected (n=2) neonates and neonates infected with wt (n=5) or Δ sopB (n=4) S . Typhimurium at day 2 p.i.. White boxes indicate enlarged images (i, ii). Counterstaining with DAPI (blue). Bar=50 µm; insert, 20 µm. (G) Number of TUNEL positive cells in tissue sections of uninfected (n=2) neonates and the neonates infected with wt (n=5) or Δ sopB (n=4) S . Typhimurium at day 2 p.i. 9 -12 images with the size of 624µm x 501µm were evaluated per animal. (H) Ki67 (red) immunostaining on small intestine tissue sections of the neonates infected with wt (n=3) or Δ sopB (n=4) S . Typhimurium at day 2 p.i.. Counterstaining with E-cadherin (green), WGA (white), and DAPI (blue). Bar=50 µm, insert: 20 µm. (I) Number of Ki67 positive cells in small intestinal tissue sections of uninfected (n=2) neonates and the neonates infected with wt (n=5) or Δ sopB (n=4) S . Typhimurium at day 2 p.i. 5-29 crypts were analyzed per section. Representative images are shown. Quantified data are shown as individual points in violin plots, solid lines represent the median. Data were analyzed using One-way ANOVA Kruskal–Wallis test with Dunn’s multiple comparisons post-test. ns, non-significant; *, p<0.05; ****, p<0.0001.

Article Snippet: Rabbit anti-Ki67 (Ab15580, Abcam), mouse anti-E-cadherin (610182, BD Transduction Laboratories), rat anti-PMN (Ly6-6B2, SeroTec), rabbit anti-Muc2 (GTX100664, BIOZOL) were used at the appropriate dilution followed by the fluorophore-conjugated donkey secondary antibody (Jackson ImmunoResearch).

Techniques: Staining, Infection, Immunostaining, TUNEL Assay