Journal: bioRxiv

Article Title: Mmp17- deficient mice exhibit heightened goblet cell effector expression in the colon and increased resistance to chronic Trichuris muris infection

doi: 10.1101/2023.07.10.548379

Figure Lengend Snippet: ( a ) Graphs of RNAseq data of naive mouse colon crypts from WT ( +/+ ) and Mmp17 KO ( -/- ) mice depicting the expression levels of Ang4 , Clca1, Retnlb and Muc2 , n=3, padj calculated with DESeq2, * p<0.05, ** p<0.01, *** p<0.001. ( b ) Immunofluorescence (IF) images of Colon Swiss rolls of naive mouse full-length colon of WT ( +/+ ) and Mmp17 KO ( -/- ) mice, for RELM-β (green), nuclear stain (blue), proximal end on the outside, scale 1 mm. ( c ) IF stain images of naive mouse proximal colon of WT ( +/+ ) and KO ( -/- ) mice for CLCA1 (green), nuclear stain (blue), scale 100 µm. ( d ) The pictures on the left show Alcian blue stain for goblet cells, on the right is a count of goblets/crypt in proximal colon of naive WT ( +/+ ) (n=4) and KO ( -/- ) (n=3) mice, 30 crypts counted for each animal. ( e ) Graphs of RNAseq data of naive mouse colon crypts from Mmp17 +/+ and -/- mice showing the expression for Atoh1 and Spdef , n=3, padj * p<0.05, ** p<0.01, *** p<0.001. ( f ) Graph of qPCR data for Retnlb of WT ( +/+ ) and KO ( -/- ) mouse colonoids after one split, Ct value relative to HPRT, n=4. ( g ) IF staining images for RELM-β in WT ( +/+ ) and KO ( -/- ) mouse colonoids after one split, RELM-β (green) and UEA-1 (magenta), scale 50 µm. Numerical data are means ± SEM. Data in (a) and (e) represents p-adjusted value from RNAseq analysis, and data in (d) and (f) was analyzed using Student’s unpaired t-test (two-tailed).

Article Snippet: The following anti-mouse primary antibodies were used: anti-RELM-beta (1:500, rabbit polyclonal antibody, Peprotech, 500-p215), anti-MUC2 (1:200, Santa Cruz Biotechnology, sc-15334), anti-CLCA1 (1:200, rabbit monoclonal antibody, Abcam, ab180851), anti-CD68 (1:200, BioRad, MCA1957T), anti-Ki67 (1:200, rabbit monoclonal antibody, Invitrogen, MA5-14520) anti-SMAD4 (for frozen sections, dilution 1:400, Cell signaling, 46535), anti-βGalactosidase (for frozen sections, dilution 1:100, Rabbit polyclonal antibody, Abcam, ab4761), anti-PDGFR-alpha (15 µg/mL, goat polyclonal antibody, R&D systems, AF1062).

Techniques: Expressing, Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Mmp17- deficient mice exhibit heightened goblet cell effector expression in the colon and increased resistance to chronic Trichuris muris infection

doi: 10.1101/2023.07.10.548379

Figure Lengend Snippet: ( a ) Muc2 IF and UEA-1 stain of naive mouse cecum of WT ( +/+ ) and KO ( -/- ) mice, Muc2 (green), UEA-1 (magenta), nuclear stain (blue), scale bar 100 µm. ( b ) Count of goblet cells per cecum crypts of naive WT ( +/+ ) and KO ( -/- ) mice, n=3. ( c ) The graph shows the measurement of goblet cell diameter in lower and upper cecum crypts of naive WT ( +/+ ) and KO ( -/- ) mice, n=3, 40-50 cells measured. ( d ) RELM-beta IF stain of naive mouse cecum of WT ( +/+ ) and KO ( -/- ) mice, RELM-beta (green), nuclear stain (blue), scale bar 100 µm (right) and count of RELM-beta positive cells per crypt, n=3, min 32 crypts counted (left). ( e ) CLCA1 IF stain of naive mouse cecum of WT ( +/+ ) and KO ( -/- ) mice, CLCA1 (green), nuclear stain (blue), scale bar 100 µm (right) and count of CLCA1 positive cells in the lower crypt, n=3, 35 crypts counted (left). Numerical data are means ± SEM. Data was analyzed using Student’s unpaired t-test (two-tailed) (b,d,e) and 2way ANOVA (c).

Article Snippet: The following anti-mouse primary antibodies were used: anti-RELM-beta (1:500, rabbit polyclonal antibody, Peprotech, 500-p215), anti-MUC2 (1:200, Santa Cruz Biotechnology, sc-15334), anti-CLCA1 (1:200, rabbit monoclonal antibody, Abcam, ab180851), anti-CD68 (1:200, BioRad, MCA1957T), anti-Ki67 (1:200, rabbit monoclonal antibody, Invitrogen, MA5-14520) anti-SMAD4 (for frozen sections, dilution 1:400, Cell signaling, 46535), anti-βGalactosidase (for frozen sections, dilution 1:100, Rabbit polyclonal antibody, Abcam, ab4761), anti-PDGFR-alpha (15 µg/mL, goat polyclonal antibody, R&D systems, AF1062).

Techniques: Staining, Two Tailed Test

Journal: International Journal of Genomics

Article Title: An Analysis of the Gene Expression Associated with Lymph Node Metastasis in Colorectal Cancer

doi: 10.1155/2023/9942663

Figure Lengend Snippet: TMEM59L, CLCA1, and TUBB2B expression in colorectal cancer. (a) IHC staining of TMEM59L, CLCA1, and TUBB2B proteins in human rectal cancer tissues (200×) (N0: non-lymph node metastasis tissues; N1–N2: lymph node metastasis tissues). (b) IHC staining of TMEM59L, CLCA1, and TUBB2B proteins in human colon cancer tissues (200×) (N0: non-lymph node metastasis tissues; N1–N2: lymph node metastasis tissues).

Article Snippet: Sections were stained with TMEM59L antibody (ab105417, 2.5 μ g/ml, Abcam, Cambridge, UK), calcium-activated chloride channel regulator 1 (CLCA1) antibody (MAB10766, 5 μ g/ml, R&D systems, Minneapolis, MN, USA), TUBB2B antibody (sc-47751, 5 μ g/ml, Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Immunohistochemistry

Journal: EBioMedicine

Article Title: Calcium-activated Chloride Channel Regulator 1 (CLCA1) Controls Mucus Expansion in Colon by Proteolytic Activity

doi: 10.1016/j.ebiom.2018.05.031

Figure Lengend Snippet: CLCA1 is a candidate to be the metalloprotease involved in mucus growth in WT and GF colon. (a) Distal colon mucus growth rate measured in Conv-R and GF mice without (−) and with (+) 10 mM EDTA applied apically ( n = 4–6). (b) Proteome analysis of mouse and human colonic mucus. The five shared most abundant proteins are labeled with their gene names. CLCA1 is marked in red (mouse n = 7, human n = 47). (c) Schematic representation of CLCA1 with marked domains. (d) Immunostainings of CLCA1 (red) and MUC2 (green) in Conv-R and GF mice, and in human sigmoid colon sections. DNA was stained with Hoechst (blue) (representative of 9–15). Scale bar is 100 μm. Data is presented as mean ± SEM; * p ≤ .05, ** p ≤ .01, with two-way ANOVA with Sidak's multiple comparisons test (MCT).

Article Snippet: Immunohistochemistry was performed on dewaxed, rehydrated and antigen retrieved sections using mouse monoclonal anti-CLCA1 (ab129283, Abcam, RRID: AB_11155317 ) and rabbit polyclonal anti-MUC2 C3 primary antibodies [ ].

Techniques: Labeling, Staining

Journal: EBioMedicine

Article Title: Calcium-activated Chloride Channel Regulator 1 (CLCA1) Controls Mucus Expansion in Colon by Proteolytic Activity

doi: 10.1016/j.ebiom.2018.05.031

Figure Lengend Snippet: CLCA1 causes increased mucus growth and penetrability in WT and Clca1 −/− mice. (a) Distal colon mucus growth rate measured in WT and Clca1 −/− mice without (−) and with (+) 5 μg rCLCA1 applied apically (n = 4–7). (b) Representative z-stack projections of bacteria sized bead (1 μm, red) distribution in distal colon mucus of WT and Clca1 −/− at 30 min after control (PBS) treatment (left panel), or after rCLCA1 (10 μg) treatment (right panel) ( n = 8). Dashed line indicates mucus surface. (c) Distribution of beads in control or rCLCA1 (10 μg) treated Clca1 −/− mucus. The z-position with highest bead intensity was defined as the mucus surface, and the bead penetrance in the mucus was plotted as % of maximum intensity at each z-position below the mucus surface ( n = 4). (d) Area under curve (AUC) analysis of bead penetrability data (c) comparing rCLCA1 treated with control mucus (n = 4). (e) Change in average bead speed in different z-planes above the mucus surface between t = 20 min and t = 60 min in control or rCLCA1 (10 μg) treated Clca1 −/− mucus, where the treatments were applied at t = 30 min. Bead mobility data is shown in , ( n = 3–5). Data is presented as mean ± SEM; * p ≤ .05, ** p ≤ .01, *** p ≤ .001 with either two-way ANOVA with Tukey's MCT (a) or unpaired 2-tailed t -test (d). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) CLCA1 causes increased mucus growth and penetrability in WT and Clca1 −/− mice. (a) Distal colon mucus growth rate measured in WT and Clca1 −/− mice without (−) and with (+) 5 μg rCLCA1 applied apically (n = 4–7). (b) Representative z-stack projections of bacteria sized bead (1 μm, red) distribution in distal colon mucus of WT and Clca1 −/− at 30 min after control (PBS) treatment (left panel), or after rCLCA1 (10 μg) treatment (right panel) ( n = 8). Dashed line indicates mucus surface. (c) Distribution of beads in control or rCLCA1 (10 μg) treated Clca1 −/− mucus. The z-position with highest bead intensity was defined as the mucus surface, and the bead penetrance in the mucus was plotted as % of maximum intensity at each z-position below the mucus surface ( n = 4). (d) Area under curve (AUC) analysis of bead penetrability data (c) comparing rCLCA1 treated with control mucus (n = 4). (e) Change in average bead speed in different z-planes above the mucus surface between t = 20 min and t = 60 min in control or rCLCA1 (10 μg) treated Clca1 −/− mucus, where the treatments were applied at t = 30 min. Bead mobility data is shown in Videos S1 and S2 ( n = 3–5). Data is presented as mean ± SEM; * p ≤ .05, ** p ≤ .01, *** p ≤ .001 with either two-way ANOVA with Tukey's MCT (a) or unpaired 2-tailed t -test (d).

Article Snippet: Immunohistochemistry was performed on dewaxed, rehydrated and antigen retrieved sections using mouse monoclonal anti-CLCA1 (ab129283, Abcam, RRID: AB_11155317 ) and rabbit polyclonal anti-MUC2 C3 primary antibodies [ ].

Techniques:

Journal: EBioMedicine

Article Title: Calcium-activated Chloride Channel Regulator 1 (CLCA1) Controls Mucus Expansion in Colon by Proteolytic Activity

doi: 10.1016/j.ebiom.2018.05.031

Figure Lengend Snippet: CLCA1 acts as a metalloprotease to expand mucus. (a) The effect of rCLCA1 (5 μg) in combination with different protease inhibitors in Clca1 −/− mucus presented as delta growth rate (growth rate of inhibitor + rCLCA treated mucus subtracted by the growth rate of inhibitor only treated mucus). Untreated and rCLCA1 alone treated mucus are also shown. cOmpl 1×, EDTA 10 mM, EGTA 10 mM, TPEN 1 mM, batimastat (Batim.) 200 μM, and bestatin (Best.) 130 μM ( n = 3–8). (b) Mucus growth rate in untreated, rCLCA1 (5 μg) or EDTA (10 mM) treated human sigmoid colon biopsies (n = 3–4). (c) The metallohydrolase motif and the cleavage site are indicated on a schematic representation of CLCA1. (d) Western blot analysis of recombinantly expressed and purified CLCA1 with unmodified sequence (WT), p.E157Q protease null mutation (E157Q) and p.GALY694-697DFRE (DFRE) cleavage site mutation (representative of 4–7). FL = full-length CLCA1, N = N-terminal CLCA1 product. (e) Effect of rCLCA1 mutants (5 μg) on Clca1 −/− mucus growth rate expressed as delta growth rate. Ctrl = control treated (PBS), WT = unmodified rCLCA1, E157Q = protease null p.E157Q mutant, DFRE = p.GALY694-697DFRE cleavage site mutant (n = 4–7). (f) Representative (of 5) western blot analysis of CLCA1 in human mucus and from wild-type (WT) and Clca1 −/− mouse mucus. FL = full-length CLCA1, N = N-terminal CLCA1 product, X = 50 kDa N-terminal product of CLCA1. Data is presented as mean ± SEM; ns = non-significant, # or * p ≤ .05, ** p ≤ .01 as determined with one-way ANOVA followed by MCT (a: Dunnett's MCT vs only rCLCA1 treatment, b: Dunnett's MCT vs untreated, e: Tukey's MCT, * and # denote significance vs Ctrl and WT respectively).

Article Snippet: Immunohistochemistry was performed on dewaxed, rehydrated and antigen retrieved sections using mouse monoclonal anti-CLCA1 (ab129283, Abcam, RRID: AB_11155317 ) and rabbit polyclonal anti-MUC2 C3 primary antibodies [ ].

Techniques: Western Blot, Purification, Sequencing, Mutagenesis

Journal: EBioMedicine

Article Title: Calcium-activated Chloride Channel Regulator 1 (CLCA1) Controls Mucus Expansion in Colon by Proteolytic Activity

doi: 10.1016/j.ebiom.2018.05.031

Figure Lengend Snippet: CLCA1 does not mediate its effect by mucus or ion secretion. (a) Mucus growth rate measured in untreated, rCLCA1 (5 μg), and CCh (1 mM) treated mucus from Clca1 −/− mice ( n = 5–7). (b) Transepithelial potential difference (PD) was recorded in untreated (Ctrl, red), rCLCA1 (purple), and CCh (green) treated Clca1 −/− tissues. Treatments were added at t = 32 min (arrow) (n = 4). (c) Representative AB-PAS stained sections from untreated (Ctrl), rCLCA1, and CCh treated Clca1 −/− tissues. Dilated crypt bottoms (filled arrowhead) and emptied goblet cells at the crypt opening (open arrowhead) are indicated in CCh treated tissues. Scale bars are 100 μm (n = 5–7). (d) The effect of rCLCA1 (5 μg) in combination with different ion channel inhibitors on the mucus presented as delta growth rate (growth rate of inhibitor + rCLCA1 treated mucus subtracted by the average growth rate of inhibitor only treated mucus). Values for untreated and rCLCA1 alone treated mucus are also shown. CaCCinh A01 (A01) 100 μM, DIDS 200 μM, and NFA 300 μM. N = 4–5. Denoted significances vs only rCLCA1 treatment. Data is presented as mean ± SEM; ns = non-significant, * p ≤ .05, ** p ≤ .01 with one-way ANOVA with Tukey's MCT.

Article Snippet: Immunohistochemistry was performed on dewaxed, rehydrated and antigen retrieved sections using mouse monoclonal anti-CLCA1 (ab129283, Abcam, RRID: AB_11155317 ) and rabbit polyclonal anti-MUC2 C3 primary antibodies [ ].

Techniques: Staining

Journal: EBioMedicine

Article Title: Calcium-activated Chloride Channel Regulator 1 (CLCA1) Controls Mucus Expansion in Colon by Proteolytic Activity

doi: 10.1016/j.ebiom.2018.05.031

Figure Lengend Snippet: The effect of CLCA1 on the mucus structure. (a) The effect of different classes of protease inhibitors on WT (blue) and Clca1 −/− (red) mucus growth rate. EDTA 10 mM, cOmpl 1×, E64-c 20 μM (n = 3–5). * denotes significance to Ctrl within each genotype, or as indicated by brackets. (b) Flushed distal colon explants from WT and Clca1 −/− mice were stained with fluorescent UEA1 lectin (red) to visualize the mucus. Images were acquired by confocal microscopy in z-stacks represented by x/y-axis (upper panels) and x/z-axis (lower panels). White arrows indicate axis direction; dashed lines indicate inner mucus (IM). (c) Magnified view of the mucus surface from ex vivo UEA1 lectin (grey) stained colonic mucus from WT and Clca1 −/− mice. (d) Mucus on flushed tissue stained with UEA1 were used to map isosurfaces as a measure of clumped mucus. (e) Detection of clumped mucus was quantified by mapping isosurfaces to a threshold level of UEA1 fluorescence. (f) Images of unflushed explants stained with UEA1 to detect mucus and Syto-9 to detect mucosal bacteria on WT and Clca1 −/− mouse colon explants. White lines indicate tissue surface; white arrows indicate the interface between the inner (IM) and outer (OM) mucus layers. Right panels show magnified images of mucosal bacteria (yellow boxes) and the IM/OM interface (blue boxes). Data is presented as mean ± SEM of 3–5 (a) or 4 per group (b-f) animals; ns = non-significant, *p ≤ .05, ****p ≤ .0001 as determined with two-way ANOVA followed by Tukey's MCT (A) or by Mann-Whitney test (e). All figure scale bars are 50 μm.

Article Snippet: Immunohistochemistry was performed on dewaxed, rehydrated and antigen retrieved sections using mouse monoclonal anti-CLCA1 (ab129283, Abcam, RRID: AB_11155317 ) and rabbit polyclonal anti-MUC2 C3 primary antibodies [ ].

Techniques: Staining, Confocal Microscopy, Ex Vivo, Fluorescence, MANN-WHITNEY

Journal: Discover oncology

Article Title: Disulfidptosis-based molecular clustering and prognostic signatures predict patient survival and the immune landscape in patients with colon cancer.

doi: 10.1007/s12672-025-02142-w

Figure Lengend Snippet: Fig. 5 Prognostic and immune cell correlation analy- sis of PRDEGs. a–j KM curves were independently analyzed at the transcriptome level to determine the prognostic value of these PRDEGs (a–j: HEYL, FSTL3, HOMER3, CALB2, FABP4, MEGF6, ADAM8, TIMP1, CLCA1, NOS2). k The results of the correlation anal- ysis between the PRDEGs and each infiltrating immunocyte are displayed in a heatmap

Article Snippet: Primary antibody information for the PRDEGs was as follows: HEYL (GB114972, Servicebio, 1:1000), NOS2 (GB11119, Servicebio, 1:500), FABP4 (GB115466, Servicebio, 1:200), TIMP1 (GB14158, Servicebio, 1:100), MEGF6 (bs-18776R, Bioss, 1:200), FSTL3 (A10279, abclonal, 1:200), CALB2 (66496-1-Ig, Proteintech, 1:4000), HOMER3 (16624-1-AP, Proteintech, 1:200), ADAM8 (23778-1-AP, Proteintech, 1:50), and CLCA1 (A15041, abclonal, 1:200).

Techniques:

Journal: Discover oncology

Article Title: Disulfidptosis-based molecular clustering and prognostic signatures predict patient survival and the immune landscape in patients with colon cancer.

doi: 10.1007/s12672-025-02142-w

Figure Lengend Snippet: Fig. 10 Immunohistochemi- cal images (ADAM8, CLCA1, FABP4, FSTL3, HOMER3)

Article Snippet: Primary antibody information for the PRDEGs was as follows: HEYL (GB114972, Servicebio, 1:1000), NOS2 (GB11119, Servicebio, 1:500), FABP4 (GB115466, Servicebio, 1:200), TIMP1 (GB14158, Servicebio, 1:100), MEGF6 (bs-18776R, Bioss, 1:200), FSTL3 (A10279, abclonal, 1:200), CALB2 (66496-1-Ig, Proteintech, 1:4000), HOMER3 (16624-1-AP, Proteintech, 1:200), ADAM8 (23778-1-AP, Proteintech, 1:50), and CLCA1 (A15041, abclonal, 1:200).

Techniques:

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: MUC2 and FCGBP expression are coordinately regulated in vitro . (A) Temporal mRNA expression of MUC2 and FCGBP in WT and CRISPR-Cas9 gene-edited MUC2 KO human LS174T goblet-like cells in culture. mRNA was isolated from cells daily for 9 days (n=8). ** p < 0.01, **** p < 0.0001. (B) Temporal MUC2 and FCGBP protein expression in LS174T goblet-like cells in culture. Protein was quantified every 2 days. GAPDH was used as a loading control. SG = stacking gel. Gly MUC2 = glycosylated MUC2 antibody (n=5). (C) mRNA expression of MUC2 and FCGBP in LS174T goblet-like cells basally and in response to PMA. qPCR was performed to quantify the most abundant mRNA transcripts for MUC2 and FCGBP and both were upregulated in response to the mucus secretagogue PMA in WT cells (n=10). **** p < 0.0001. (D) PMA upregulated protein expression of both MUC2 and FCGBP in LS174T goblet-like cells. Cell lysates were analyzed by SDS-PAGE/Western blot with antibodies against FCGBP and MUC2. GAPDH was used as a loading control. SG = stacking gel (n=5). (E) Workflow for proteomics analysis to compare WT and MUC2 KO cell lysates. WT cells were tagged with a light dimethylation tag, and MUC2 KO cells were tagged with a heavy dimethylation tag. Both cells were run through the mass spectrometer, and the results were run through MaxQuant and Metascape. (F) Metascape analysis showing gene ontology enrichment analysis associated with WT and MUC2 KO cells. (G) Metascape analysis showing gene ontology biological processes utilized by WT and MUC2 KO cells. Pathways of interest are highlighted in red.

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Expressing, In Vitro, CRISPR, Isolation, SDS Page, Western Blot, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: The most abundant proteins that were up- and down-regulated between WT and MUC2 KO LS174T goblet cells by proteomics analysis.

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Migration

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: MUC2 and FCGBP localization within goblet-like cells. (A) Confocal microscopy images of WT and MUC2 KO LS174T cells showing MUC2 (red), FCGBP (green), and DAPI (blue). Note FCGBP bound to MUC2 and free FCGBP in the cytoplasm of goblet-like cells. Images are representative of 5 different experiments. Scale bar = 10 μm. (B) Quantification of colocalization between MUC2 and FCGBP in WT cells by confocal microscopy images. ***p<0.001 (n=15). (C) Mean fluorescence intensity (MFI) of FCGBP and (D) MUC2 compared to WT cells of confocal images taken in (A) ***p<0.001 (n=15).

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Confocal Microscopy, Fluorescence

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: MUC2 and FCGBP expression are coordinately regulated in animals. (A) Colonic mouse organoids were isolated and grown basally or in the presence of DAPT to skew cells to a goblet cell phenotype and mRNA expression was measured through RT-PCR (n=5-6). (B) Fcgbp protein expression in colonic organoids using Western blot analysis. β-Actin was used as a loading control (n=4). (C) mRNA expression from proximal and distal colon tissues of Muc2 +/+ and Muc2 -/- littermates. ****p<0.0001 (n=5-10 mice per group). (D) Representative Western blot of the expression of Fcgbp and Muc2 in mouse tissues from the proximal and distal colon. β-Actin was used as a loading control (n-5-10). (E) mRNA from colonic tissues of SPF mice, antibiotics-treated SPF mice, or GF mice were compared for Muc2 and Fcgbp expression through RT-PCR analysis. *p<0.05, ** p < 0.01. SPF, Specific pathogen free; Abx, antibiotics; GF, Germ free; (n=5-10 mice per group). (F) Protein expression of Fcgbp and Muc2 in SPF, Abx, and GF proximal colonic tissue through Western blotting analysis. β-Actin was used as a loading control (n=5).

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: Proteomics analysis of mucus associated proteins found in mouse colonic tissues and their hit number.

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques:

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: FCGBP is highly enriched in mucin granules from goblet-like cells. (A) Western blot analysis of LS174T mucin granules purified by differential centrifugation and sucrose gradient ultracentrifugation. MUC2 and FCGBP were localized in high-density fraction 9. SG = stacking gel. (B) Goblet cell mucin granules from A above were visualized by transmission electron microscopy highlighting the lipid bilayer. Inset is a close-up view of the lipid bilayer. Scale bar (top right) = 100 nm. (C) Mucin granules treated with weak detergents CHAPS and octyl-glucoside to solubilize the lipid bilayer showed increased immunoreactivity of FCGBP and MUC2. SG = stacking gel. (D) Treatment of LS174T goblet cell with the N-linked glycosylation inhibitor tunicamycin caused a shift in MUC2 apoprotein and FCGBP migration pattern on a 1% agarose gel or SDS-PAGE/Western blot, as well as a marked decreased in glycosylated MUC2. ***p<0.001. (E) Widefield microscopy images of purified mucin granules stained for MUC2 (red) or FCGBP (green) demonstrating colocalized of both proteins (arrows). Images are representative of 3 separate experiments. Scale bar = 20 μm. (F) Quantification of the number of MUC2 positive granules from E that contain FCGBP (n=10). (G) STED images of granules isolated from LS174T goblet-like cells stained for MUC2 (red) and FCGBP (green). Images are representative of 3 separate experiments. Scale bar = 0.5 μm. (H) Quantification of MUC2 and FCGBP in granules imaged through STED imaging. (I) Quantification of colocalization between MUC2 and FCGBP imaged through STED imaging. 24 unique images were used from 3 separate granule preparations.

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Western Blot, Purification, Centrifugation, Transmission Assay, Electron Microscopy, Migration, Agarose Gel Electrophoresis, SDS Page, Microscopy, Staining, Isolation, Imaging

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: Proteomic analysis of purified human mucin granules from LS174T cells under reducing and non-reducing conditions. (A) Shotgun proteomic analysis workflow. (B) Western blot of mucin granules run on SDS-PAGE under reducing and non-reducing conditions and probed with an antibody for glycosylated MUC2. The red-boxed areas designated as fractions F1 to F4 were excised for proteomic analysis. MUC2/MAP (mucus-associated protein) protein of interest for each fraction is shown on the right-hand side of each box. Bold red arrowheads indicate the junction between the stacking gel (SG) and the resolving gel. (C) Metascape analysis showing gene ontology enrichment of pathways utilized by each fraction under reducing conditions. Pathways of interest are highlighted in red. (D) Metascape analysis showing gene ontology biological processes associated with the proteins present in each of the 4 fractions under reducing conditions. Pathways of interest are highlighted in red. (E) Metascape analysis showing transcription factors associated with protein hits from each mucin granule fraction under reducing conditions. Transcription factors of interest are highlighted in red. (F) Venn diagram showing overlapping and unique proteins from each of the 4 fractions. The Venn diagram was made with Venny . (G) STRING analysis showing known protein-protein interactions for FCGBP and MUC2.

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Purification, Western Blot, SDS Page

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: Attractive protein hits from the proteomic analysis of purified mucin granules derived from human LS174T goblet cells.

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Purification, Derivative Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: FCGBP is found in secreted mucin from LS174T goblet-like cells and is non-covalently attached to MUC2. (A) Sepharose 4B (S4B) column chromatography of native LS174T secreted mucus. The colored sections represent different molecular weight fractions from S4B and were probed using Western blotting for MUC2 and FCGBP. Arrows indicate the location of known molecular weight standards: Blue Dextran (BD: >2x10 6 KDa), Thyroglobulin (TG: 669 KDa), Bovine Serum Albumin (BSA: 67 KDa), and Chymotrypsinogen A (CTA: 25 KDa). (B) S4B fractions ran under reducing and non-reducing conditions for Western blot analysis. Blots were immunoblotted with antibodies for FCGBP, MUC2 glycosylated proteins, and CLCA1. FCGBP separated from MUC2 under reducing conditions but remains intact in the V 0 stacking gel (SG) under non-reducing conditions. Major protein bands of interest are highlighted in red. The red arrowheads indicate the junction between the stacking gel (SG) from the running gel. SDS-PAGE gels probed for FCGBP consisted of 4% stacking gel and 12% running gel and for MUC2 and CLCA1, 4% stacking gel and 7.5% running gel. (C) CsCl density ultracentrifugation of MUC2 V o mucin isolated by S4B in A above. The location of the low-density fraction where FCGBP partitioned and the high-density fractions that contain MUC2 are boxed in red. (D) Western blot analysis for FCGBP and MUC2 for each of the CsCl density ultracentrifugation fractions. Note that MUC2 is present in the high-density fractions 5-8, whereas FCGBP is only present in the lower-density fraction 4. SG, stacking gel.

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Column Chromatography, Molecular Weight, Western Blot, SDS Page, Isolation

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: Immunolocalization of MUC2 and FCGBP in secreted mucus stands from LS174T goblet-like cells. (A) STED imaging of goblet-like cells secreting MUC2 mucin (red) and FCGBP (green) showing granule release and the polymerization of MUC2 and FCGBP towards the tail region. Scale bar = 1 μm. Images are representative of 3 independent experiments. (B) Quantification of total MUC2 and FCGBP in secreted mucin through STED imaging. (C) Quantification of MUC2 and FCGBP in the flare, mid, and tail sections of the mucus strand. Images are representative of 3 independent experiments. ** p < 0.01. (D) STED imaging of LS174T cells showing the fate of MUC2 and FCGBP in mucus strands. Scale bar = 1 μm. Images are representative of 3 independent experiments.

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: MUC2 and FCGBP expression during restitution in LS174T WT and MUC2KO goblet-like cells. (A) WT and MUC2KO cells were grown with a wound insert leaving a gap of 500 μm. At 0 to 7 days post-wound, the space between the cells was measured to quantify restitution. *p<0.05, ****p<0.0001 (n=6 per day). (B) Representative bright field images of the restitution assay. The solid black dotted lines represent the cell front during wound healing. (C) Confocal microscopy images of control WT and MUC2KO during restitution showing MUC2 (red), FCGBP (green), and DAPI (blue). The open arrows show the direction of cell migration. The stippled white boxed area shows the wound margin and outside of the wound margin that was quantified to enumerate partitioning of MUC2 and FCGBP during wound healing. Images are representative of 3 different experiments. (D) At 0 to 7 days post-wound, MUC2, FCGBP, and ATF4 mRNA were analyzed by RT-PCR. GAPDH was used as a housekeeping gene. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (n=6 per day).

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Expressing, Confocal Microscopy, Migration, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing

doi: 10.3389/fimmu.2023.1211336

Figure Lengend Snippet: Fcgbp expression is altered in DSS colitis in mice. (A) Weight loss in Muc2 +/+ (blue) and Muc2 -/- (red) littermates weighed daily after exposure to DSS in their drinking water during acute disease and in restitution. Shown are the percent weight loss/gain from day 0 (n=8-12 mice per day). (B) Muc2 and Fcgbp mRNA expression during the 15-day duration of the experiment. β-Actin was used as a housekeeping gene. *p<0.05, ***p<0.001, ****p<0.0001 (n=8-12). (C) Fcgbp protein expression in colonic tissues taken during the 15-day duration of the experiment. β-Actin was used as a housekeeping gene. (D) Quantification of protein from Western blot analysis during the 15-day duration of the experiment. Protein levels were compared to their own control (day 0). ****p<0.0001 (n=8-12). (E) Math1 and Spdef mRNA expression during the 15-day duration of the experiment. β-Actin was used as a housekeeping gene. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (n=8-12).

Article Snippet: The membranes were then probed with primary antibodies: MUC2 (in-house antibody against CsCl purified glycosylated MUC2 mucin ( ) or Santa Cruz sc-515106 for the C-terminus), FCGBP (Cloud-Clone PAP389Hu01), and CLCA1 (Abcam 1808521).

Techniques: Expressing, Western Blot