mouse anti muc6  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Single Cell Integration Characterises Gastric Cell Metaplasia in Inflammatory Intestinal Diseases

doi: 10.1101/2024.10.11.614415

Figure Lengend Snippet: a) Differential abundance analysis of cell neighbourhoods from Martin 2019 dataset based on embedding on the whole atlas. Cell neighbourhoods with positive log fold change are enriched in CD compared to healthy samples. b) Overview of the number of MGN (Mucous gland neck)/INFLAREs ( Infla mmatory E pithelial cell s ) and donors per study, broken down by age and region of the GI. Dot size indicates the number of donors, colour indicates the number of cells. c) Subclustering of surface foveolar cells, showing heterogeneity of marker genes and additional genes upregulated in metaplastic surface foveolar cells. d) Deconvolution (BayesPrism) of bulk RNAseq dataset comparing MGN/INFLAREs in healthy (normal, n = 50) and CD (n = 254). e) Deconvolution (BayesPrism) of TCGA bulk RNAseq data of MGN/INFLAREs in healthy tissue (normal, n = 41) and tumour tissue stratified by microinstability status, n = 40 (Tumour_MSI-H), n = 42 (Tumour_MSI-L), n = 126 (Tumour_MSS) and n = 272 (Tumour_NA). MSI-high tumours are predicted to have higher levels of INFLAREs. f) Deconvolution (BayesPrism) of bulk RNAseq from celiac disease comparing MGN/INFLARE proportions in healthy and celiac disease tissue. For GSE131705, n = 21 (healthy) and n = 33 (celiac). For GSE145358, n = 6 (healthy), n = 15 (celiac gluten free) and n = 15 (celiac gluten challenge). For all box and whisker plots the lower edge, upper edge and centre of the box represent the 25th (Q1) percentile, 75th (Q3) percentile and the median, respectively. The interquartile range (IQR) is Q3 - Q1. Outliers are values beyond the whiskers (upper, Q3 + 1.5 x IQR; lower, Q1 - 1.5 x IQR). g) Protein and ABPAS staining of INFLAREs (MUC6, Magenta+Blue+ ABPAS staining) and Surface foveolar cells (MUC5AC) in CD ileum showing association with tertiary lymphoid structures (dense nuclei and CD3/CD20+ regions). h) Protein staining of INFLAREs (MUC6) and Surface foveolar cells (MUC5AC) from CD ileum tissue from additional donors. i) smFISH staining of INFLARE ( Infla mmatory E pithelial cell) markers ( MUC6, AQP5 and BPIFB1 ) in pyloric metaplasia of CD duodenum showing heterogeneity in AQP5 and BPIFB1 expression. j) Protein staining of INFLAREs (MUC6) in celiac disease duodenum from two separate donors. k) Protein staining of INFLAREs (MUC6) and Surface foveolar cells (MUC5AC) in colon resection tissue from ulcerative colitis patients. Upper and lower panels are images from two different patients.

Article Snippet: IHC was conducted for the following commercially available antibodies: anti-mouse MUC5AC (Sigma-Aldrich, MAB2011, 1:4000) and anti-mouse MUC6 (ScyTek, RA0224-C.1, 1:4000).

Techniques: Marker, Whisker Assay, Staining, Expressing

Journal: bioRxiv

Article Title: Single Cell Integration Characterises Gastric Cell Metaplasia in Inflammatory Intestinal Diseases

doi: 10.1101/2024.10.11.614415

Figure Lengend Snippet: a) UMAP showing cells from small intestinal epithelium in the full atlas (healthy and diseased). MGN (mucous gland neck)/INFLARE and surface foveolar cells, both involved in pyloric mucous gland metaplasia, are highlighted with a dashed circle. b) Marker gene dot plot of gastric mucous/pyloric gland cell markers (MGN and surface Foveolar cells). c) Proportion of MGN/INFLARE cells by disease category in duodenum and ileum. d) Bulk deconvolution (BayesPrism) using disease intestinal epithelium as a reference in studies of crohn’s disease (CD) and ulcerative colitis (UC). For E_MTAB_5464 n = 25 (CD), n = 27 (UC) and n = 27 (normal). For GSE111889 n = 122 (CD), n = 71 (UC) and n = 50 (normal). The lower edge, upper edge and centre of the box represent the 25th (Q1) percentile, 75th (Q3) percentile and the median, respectively. The interquartile range (IQR) is Q3 - Q1. Outliers are values beyond the whiskers (upper, Q3 + 1.5 x IQR; lower, Q1 - 1.5 x IQR). e) smFISH staining of MGN/INFLARE cell marker genes ( MUC6, AQP5, BPIFB1 ) and surface foveolar cell markers ( MUC5AC ) in duodenum biopsy from a patient with CD and pyloric metaplasia. f) Schematic of MGN/INFLARE cell distribution across the stomach and intestines and structure of pyloric mucous glands/Brunner’s glands.

Article Snippet: IHC was conducted for the following commercially available antibodies: anti-mouse MUC5AC (Sigma-Aldrich, MAB2011, 1:4000) and anti-mouse MUC6 (ScyTek, RA0224-C.1, 1:4000).

Techniques: Marker, Staining

Journal: bioRxiv

Article Title: Single Cell Integration Characterises Gastric Cell Metaplasia in Inflammatory Intestinal Diseases

doi: 10.1101/2024.10.11.614415

Figure Lengend Snippet: a) UMAP of small intestine epithelial cells coloured by pseudotime values inferred with Monocle3 analysis on epithelial cells from ileum only, highlighting trajectory from stem cells to MGN/INFLAREs. Small UMAP alongside indicates cell types. b) Expression of Stem and MGN/INFLARE markers along pseudotime, along with cell type abundance along the Stem - TA - MGN/INFLARE trajectory. c) Proliferation ( MKI67 ) and Stem ( LGR5 ) marker expression by smFISH in INFLAREs ( MUC6 ) from CD ileum and duodenum. d) Non-negative matrix factorisation analysis (Methods) of cell types from small intestine in the atlas. Violin plots showing expression of ranked genes in factors related to MGN (mucous gland neck)/INFLAREs ( Infla mmatory E pithelial cell s ) and stem cells ( LGR5 +). e) Gene rankings of genes in factors 5 (Stem cell factor) and 42 (MGN/INFLARE factor). Highlighted genes are involved in stem-cell function (blue) and MGN/INFLARE marker genes (red). f) Schematic of epithelial cell trajectories along the crypt-villus axis in the small intestine known during health (black arrows) and hypothesised in our study in inflammatory intestinal diseases (red dotted box and red arrows).

Article Snippet: IHC was conducted for the following commercially available antibodies: anti-mouse MUC5AC (Sigma-Aldrich, MAB2011, 1:4000) and anti-mouse MUC6 (ScyTek, RA0224-C.1, 1:4000).

Techniques: Expressing, Marker, Cell Function Assay

Journal: bioRxiv

Article Title: Single Cell Integration Characterises Gastric Cell Metaplasia in Inflammatory Intestinal Diseases

doi: 10.1101/2024.10.11.614415

Figure Lengend Snippet: a) Expression of genes related to mucosal barrier function in MGN (Mucous gland neck)/INFLAREs ( Infla mmatory E pithelial cell s ) and Surface foveolar cells in healthy stomach, healthy duodenum and IBD ileum. b) Violin plot showing expression of PIGR and CCL28 , genes involved in mucosal IgA response in Surface foveolar cells from healthy stomach, healthy duodenum and IBD ileum. c) Subclustered MGN/INFLARE cells from across the atlas (locations, ages and diseases). MGN/INFLARE cells from different regions and duodenum/stomach in utero occupy separate coordinates in the UMAP. d) Overlap of MGN/INFLARE marker genes from different regions. Marker genes of MGN/INFLARE cells were calculated by differential gene expression (wilcoxon rank sum test) of other stomach and small intestine epithelial cells separately for healthy adult stomach MGN, healthy adult duodenum MGN, ileum CD INFLARE and duodenum celiac disease INFLARE. Overlapping marker genes show greater similarity of INFLAREs to healthy adult stomach MGN cells, than to healthy adult duodenum MGN cells. e) GO terms from upregulated genes (wilcoxon rank sum test) in IBD INFLARE cells (CD and pediatric IBD) compared with healthy control duodenum. Highlighted pathways are inflammatory, MHC-II mediated antigen presentation and exogenous peptide antigen presentation related pathways. f) Analysis as in (e) comparing IBD INFLARE cells to healthy control stomach. Comparison to healthy control stomach did not highlight MHC-II related pathways as in the comparison to healthy control duodenum. g) Chemokine and MHC-II gene scores (see Supplementary Table 5 for gene list) comparing small intestine epithelial cells in the atlas in healthy control and disease (IBD and celiac) samples showing specificity of upregulated chemokine and MHC-II related gene expression in metaplastic gastric mucous glands, particularly in INFLAREs vs MGN cells. h) Expression of chemokines expressed by MGN/INFLARE cells, across healthy and diseased tissues. i) Expression of chemokines on metaplastic INFLARE and surface foveolar cells, specifically in diseased small intestine (pooled cells from IBD and celiac disease). j) Additional smFISH staining (as in ) of INFLAREs ( MUC6 ) association with ACKR1+ vessel in CD duodenum. k) Correlation between INFLARE cell proportions and cell types/genes from deconvolution (BayesPrism) of bulk RNAseq adult and pediatric IBD datasets using the atlas as a reference. Analysis indicates consistent correlation of EC_venous cells (ACKR1+ endothelial population) with INFLAREs, and Surface foveolar and neutrophil marker genes with INFLAREs. l) Protein expression in CD ileum of HLA-DR (MHC-II) in INFLAREs (MUC6) along with localisation of CD3+ T cells and regulatory T cells (FoxP3+CD3+). m) Expression per donor of genes involved in IFNGR to MHC-II signalling pathway in INFLAREs and MGN cells in small intestine, as summarised in . n) Additional protein staining for INFLAREs (MUC6) in CD disease ileum (as in ) with various T cell subsets (CD4+CD3+, CD8+CD3+, TCRγδ+CD3+ T cells). o) Protein staining as in (n) in Celiac disease duodenum tissue.

Article Snippet: IHC was conducted for the following commercially available antibodies: anti-mouse MUC5AC (Sigma-Aldrich, MAB2011, 1:4000) and anti-mouse MUC6 (ScyTek, RA0224-C.1, 1:4000).

Techniques: Expressing, In Utero, Marker, Control, Comparison, Staining

Journal: bioRxiv

Article Title: Single Cell Integration Characterises Gastric Cell Metaplasia in Inflammatory Intestinal Diseases

doi: 10.1101/2024.10.11.614415

Figure Lengend Snippet: a) Gene score of chemokines across MGN (Mucous gland neck)/INFLAREs ( Infla mmatory E pithelial cell s ) from stomach, duodenum and ileum across different conditions. b) Cell-cell interactions mediated by CXCL chemokines expressed by INFLAREs and various immune cells/venous endothelial cells. c) smFISH staining of INFLARE ( MUC6, BPIFB1 ), Surface foveolar ( MUC5AC ) and activated endothelial cells ( ACKR1 ) showing proximity of vessels to metaplastic glands in CD. d) Gene score of MHC-II genes and peptide processing genes across MGN/INFLARE cells from stomach, duodenum and ileum across different conditions. e) Protein staining of INFLAREs (MUC6), macrophages (CD68) and MHC-II (HLA-DR) in ileum from CD resection showing high MHC-II expression in INFLAREs. f) Schematic of signalling pathway from IFNGR to MHC-II with dotplot of gene scores from this pathway in MGN/INFLAREs from stomach, duodenum and ileum across different conditions. g) Protein staining of INFLAREs (MUC6) and CD4 T cells (CD3+CD4+), CD8 T cells (CD8+CD3+) and γδ T cells (TCRγδ+CD3+) showing interaction between CD4 T cells and INFLAREs. h) Schematic of the potential role of gastric mucous/pyloric gland metaplasia in inflammatory intestinal diseases. INFLAREs and metaplastic surface foveolar cells arise in response to local inflammation in order to promote mucosal healing. As disease progresses INFLAREs contribute to ongoing inflammation through association with activated vessels, the recruitment of various immune cells and direct interactions with CD4 T cells via MHC-II.

Article Snippet: IHC was conducted for the following commercially available antibodies: anti-mouse MUC5AC (Sigma-Aldrich, MAB2011, 1:4000) and anti-mouse MUC6 (ScyTek, RA0224-C.1, 1:4000).

Techniques: Staining, Expressing

Journal: Infection and Immunity

Article Title: Chemokines and Antimicrobial Peptides Have a cag -Dependent Early Response to Helicobacter pylori Infection in Primary Human Gastric Epithelial Cells

doi: 10.1128/IAI.01517-13

Figure Lengend Snippet: Immunodetection of CK18, MUC1, MUC5AC, and TFF1 in PGEC cells and in AGS cells. Scale bar, 50 μm.

Article Snippet: The primary mouse monoclonal Ab anti-human CK18 and MUC6 (sc-58727 and sc-33668, respectively; Santa Cruz Biotechnology) were used at 2 μg/ml in 1% donkey serum–PBS, and the primary rabbit polyclonal Ab anti-human MUC1, MUC5AC, and TFF-1 (sc-15333, sc-20118, and sc-28925, respectively; Santa Cruz Biotechnology) were used at 4 μg/ml in 1% goat serum PBS.

Techniques: Immunodetection