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: Life Science Alliance

Article Title: The arginine methyltransferase PRMT5 promotes mucosal defense in the intestine

doi: 10.26508/lsa.202302026

Figure Lengend Snippet: (A) Normalized mRNA expressions of select genes from independent pairs of WT (n = 5 or 8) and Prmt5 +/− (n = 5 or 8) colonic IECs. Each dot represents the result from one mouse. Data shown are means ± SD. * P < 0.05 ( t test). (B) Representative histograms of isotype or CLCA1 fluorescent intensity in colonic epithelial cells (ECs) from one pair of WT and Prmt5 +/− littermates. (C) Left: representative flow cytometry analysis of CLCA1 protein levels in colonic epithelial cells (Epcam + ) from two pairs of WT and Prmt5 +/− littermates. Right: summary of the % of CLCA1 + colonic epithelial cells in steady state WT and Prmt5 +/− littermates. Each dot represents the result from one mouse. Data shown are means ± SD. ** P < 0.01 ( t test). (D) Left: representative images of WGA staining of WT and Prmt5 +/− colonic sections. Right: average WGA thickness and fluorescent intensity from WT and Prmt5 +/− littermates. Each dot on the graphs represents the average measurement taken from a region of interest within one colonic section. Data shown are means ± SD. ** P < 0.01, n.s., not significant ( t test). Scale bar represents 100 μm.

Article Snippet: Fixed and permeabilized MC38 cells (Cat: 554714; BD) were stained with the anti-CLCA1 antibody for 1 h in 4°C, followed by anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody for 1 h in 4°C.

Techniques: Flow Cytometry, Staining

Journal: Life Science Alliance

Article Title: The arginine methyltransferase PRMT5 promotes mucosal defense in the intestine

doi: 10.26508/lsa.202302026

Figure Lengend Snippet: Top: representative western blots of FCGBP, MUC2, CLCA1, and βTubulin in colon IEC whole cell lysates. Bottom: quantification of FCGBP, MUC2, and CLCA1 Western blot signals from WT (n = 4) and Prmt5 +/− (n = 4–7) mice. Each dot represents the result from one mouse. Signal quantification was calculated as signal of indicated protein over those of βTubulin. Data shown are means ± SD. * P < 0.05, n.s., not significant ( t test).

Article Snippet: Fixed and permeabilized MC38 cells (Cat: 554714; BD) were stained with the anti-CLCA1 antibody for 1 h in 4°C, followed by anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody for 1 h in 4°C.

Techniques: Western Blot

Journal: Life Science Alliance

Article Title: The arginine methyltransferase PRMT5 promotes mucosal defense in the intestine

doi: 10.26508/lsa.202302026

Figure Lengend Snippet: Colons were incubated in 5 mM EDTA-containing HBSS for 20 min at 37°C to enrich for IECs (TCRβ − Epcam + ). The percentage of CLCA1-expressing IECs was quantified by FlowJo. Forward scatter is an index for cell size.

Article Snippet: Fixed and permeabilized MC38 cells (Cat: 554714; BD) were stained with the anti-CLCA1 antibody for 1 h in 4°C, followed by anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody for 1 h in 4°C.

Techniques: Incubation, Expressing

Journal: Life Science Alliance

Article Title: The arginine methyltransferase PRMT5 promotes mucosal defense in the intestine

doi: 10.26508/lsa.202302026

Figure Lengend Snippet: (A) Gating strategy for defining PRMT5 high- and low-expressors among cultured MC38 cells. Forward scatter is an index for cell size. Side scatter is an index for cell granularity. (B) Geometric mean fluorescence intensity of CLCA1 in PRMT5 high and PRMT5 low MC38 cells. Each dot represents the result from an independent experiment (n = 4). Data shown are means ± SD. * P < 0.05 ( t test). (C) Log 2 fold change of Prmt5 mRNA in MC38 cells treated with the indicated cytokine (20 ng/ml) or C. rodentium (multiplicity of infection = 20) for 12 h relative to MC38 cells treated with vehicle control (PBS for SAA1, IL-23, and C. rodentium treatments; PBS + 0.1% BSA for IL-1β treatment). Each dot represents the result from an independent experiment. Data shown are means ± SD. n.s., not significant ( t test).

Article Snippet: Fixed and permeabilized MC38 cells (Cat: 554714; BD) were stained with the anti-CLCA1 antibody for 1 h in 4°C, followed by anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody for 1 h in 4°C.

Techniques: Cell Culture, Fluorescence, Infection

Journal: Life Science Alliance

Article Title: The arginine methyltransferase PRMT5 promotes mucosal defense in the intestine

doi: 10.26508/lsa.202302026

Figure Lengend Snippet: (A) Gating strategy for assessing PRMT5 and CLCA1 protein levels in transfected (GFP high ) HEK293 ft cells. Forward scatter is an index for cell size. Side scatter as an index of cell granularity. (B) Proportion of HEK293 ft cells transfected with the empty vector control and PRMT5 WT overexpression vector (% GFP high ). Each dot represents the result from an independent experiment. Data shown are means ± SD. (C) Fold change of PRMT5 and CLCA1 gMFI in GFP high HEK293 ft cells with the PRMT5 WT overexpression vector relative to cells transduced with empty vector control (empty) 72 h post-transfection. Each dot represents the result from an independent experiment. Data shown are means ± SD. ** P < 0.01, * P < 0.05 ( t test).

Article Snippet: Fixed and permeabilized MC38 cells (Cat: 554714; BD) were stained with the anti-CLCA1 antibody for 1 h in 4°C, followed by anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody for 1 h in 4°C.

Techniques: Transfection, Plasmid Preparation, Over Expression, Transduction

Journal: Life Science Alliance

Article Title: The arginine methyltransferase PRMT5 promotes mucosal defense in the intestine

doi: 10.26508/lsa.202302026

Figure Lengend Snippet: (A) A representative Western blot result showing global symmetric dimethyl arginine and total protein in control and Prmt5 +/− colonic epithelial whole cell lysates. (B) Workflow of organoid culture from WT colonic crypt stem cells. (C) Representative fields of WT colonic organoids 48 h post-treatment with IL-13 together with 5 μM EPZ015666 or vehicle (DMSO). Scale bar represents 100 μm. (D) qRT-PCR analysis of Clca1 , Fcgbp , and Muc2 transcript levels in organoids treated with EPZ015666 for 12 h at the indicated dosage over those found in DMSO-treated controls. Results shown are average and SD from three independent experiments. ** P < 0.01, n.s., not significant ( t test).

Article Snippet: Fixed and permeabilized MC38 cells (Cat: 554714; BD) were stained with the anti-CLCA1 antibody for 1 h in 4°C, followed by anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody for 1 h in 4°C.

Techniques: Western Blot, Quantitative RT-PCR

Journal: Nature

Article Title: Colonic epithelial cell diversity in health and inflammatory bowel disease.

doi: 10.1038/s41586-019-0992-y

Figure Lengend Snippet: Fig. 4 | Heterogeneity of goblet cells in health and ulcerative colitis. a, t-SNE plot of subclusters across all captured goblet cells (n = 3 per group). b, i–iv, Newly identified and previously known goblet-cell marker proteins validated by immunohistochemistry in healthy human colonic tissue (representative of three patient samples): i, BCAS1 (red) with MUC2 (green); ii, CLCA1 (red) with MUC2 (green); iii, WFDC2 (red) with MUC2 (green); iv, REGIV (green) with MUC2 (red). v, vi, Heterogeneity in expression of these markers is also observed by double staining for WFDC2 (green) with CLCA1 (red; v) and for WFDC2 (red) with REGIV (green; vi). Scale bars, 20 μm (i), 50 μm (ii–iv, vi), 10 μm (v). c, Representative immunohistochemistry images of colon biopsies from inflamed and noninflamed regions of ulcerative colitis colon stained for WFDC2 (n = 31) and MUC2 (n = 24). The top panel shows WFDC2 and MUC2 expression in a patient with mild disease, and the bottom panel shows expression in severe inflammation. d, Quantification and distribution of change in WFDC2 and MUC2 expression (fold change (expressed in log2)) in inflamed versus noninflamed tissue from patients with varying disease severity (the 25th, 50th and 75th percentiles are shown). Each point is coloured and sized by severity score (automated quantification; see Methods). Triangles represent outliers (showing a decrease greater than 16-fold). Paired two-sided t-test: MUC2 (n = 24), P = 0.2485; WFDC2 (n = 31), P = 0.0001779. e, i, smISH of Wfdc2 in the colons of naive mice and of animals treated with DSS (a model of acute colitis) (n = 5 per group). ii, smISH quantification from colons of naive and DSS-treated mice (n = 5 per group; ∗∗∗P = 0.0008; unpaired two- sided t-test; mean ± s.e.m. shown). f, Quantification of Wfdc2 expression relative to Gapdh expression by qRT–PCR from the colons of naive and DSS-treated mice (n = 4 per group; ∗P = 0.0086; unpaired two-sided t-test; mean ± s.e.m. shown).

Article Snippet: These included; Lyz - Agilent (Dako)- A0099 (lot 20025086) (EC 3.2.1.17); WFDC2 - Novus Biologicals - Novus NBP2-46360 (clone OTI1E12) (lot W001) ; MUC2 - Agilent (Dako) - M7313 (Clone CCP58)(lot 20043026); REG4 - Atlas antibodies - HPA046555 (lot R44544); CLCA1 - Atlas Antibodies - HPA059301 (lot R82805); BCAS1 - Atlas Antibodies - HPA051816 (lot R67167); PCSK1N - Atlas Antibodies - HPA003925 (lot B106794); CPE - R&D systems - MAB3587 (clone 420709) (lot ZOG021804A) ChGA - Abcam - b15160 (lot GR244279-2); CTSE - Atlas Antibodies - HPA012940 (lot R03471), EpCAM - PeVio770(TM) / PE/Cy7 - Milteyni - 130-099-742 (lot 5171115435); CD45 APC - Milteyni - 130-098-143 (lot 5171115415); CD90 FITC - Biolegend - 328107 (clone 5E10) (lot B241989); DAPI - BD biosciences - BD564907 (lot 7089926) REG4 (IF) - R&D systems - AF1379-SP (lot IES031802C) MUC2 (IF) - ThermoFisher Scientific - MA5-12345 (lot TF258930 ) WFDC2 (IF) - Abcam - ab24480 (lot GR298303-5 ) Bestrophin 4 (IHC and FACS) - Atlas - HPA058564 (Lot R81693) Alexa Fluor 488 goat anti-rabbit IgG - Invitrogen A11034 (lot 1971948) SPINK4 (IHC) rabbit - Abcam- ab183347(lotA39120) REG1A (IHC) rat - R&D systems -Mab49371 (lot cgbr011810A) Validation Lyz - IHC - human - validation reference on supplier insert - Krugliak L, Meyer PR, Taylor CR.

Techniques: Marker, Immunohistochemistry, Expressing, Double Staining, Staining, Quantitative RT-PCR