Journal: Scientific reports

Article Title: Comprehensive bioinformatics analysis was used to identify and verify differentially expressed genes in targeted therapy of colon cancer.

doi: 10.1038/s41598-025-00011-8

Figure Lengend Snippet: Fig. 3. GSEA comparing ZG16 and high expression of CLCA1 group and low expression group. GSEA conducted on the entire genome identified enrichment items in non-differentially expressed genes, which were consistent with those identified by GO and KEGG enrichment analyses.

Article Snippet: The membranes were closed in 5% defatted milk for 2 h at room temperature and incubated with primary antibodies CLCA1 (Abconlal, CAT#A15041,1:10,000), ZG16 (Proteintech, CAT#17397-1-AP,1:5000), and β-ACTIN (Servicebio, CAT#GB11001,1:2000) overnight at 4 °C, followed by incubation with the secondary antibodies for 1 h. Finally, the indicated proteins were visualized using West Pico chemiluminescent substrate (Thermo Fisher Scientific, USA).

Techniques: Expressing

Journal: Scientific reports

Article Title: Comprehensive bioinformatics analysis was used to identify and verify differentially expressed genes in targeted therapy of colon cancer.

doi: 10.1038/s41598-025-00011-8

Figure Lengend Snippet: Fig. 5. The expression of CLCA1 and ZG16. (A) The expression of proteins in TCGA, GSE39582, and TCGA + GTEX database. (B) The density expression of CLCA1 and ZG16 in the GES39582 dataset. (C) The Top10 hotmap of DEGs in the GSE39582 dataset.

Article Snippet: The membranes were closed in 5% defatted milk for 2 h at room temperature and incubated with primary antibodies CLCA1 (Abconlal, CAT#A15041,1:10,000), ZG16 (Proteintech, CAT#17397-1-AP,1:5000), and β-ACTIN (Servicebio, CAT#GB11001,1:2000) overnight at 4 °C, followed by incubation with the secondary antibodies for 1 h. Finally, the indicated proteins were visualized using West Pico chemiluminescent substrate (Thermo Fisher Scientific, USA).

Techniques: Expressing

Journal: Scientific reports

Article Title: Comprehensive bioinformatics analysis was used to identify and verify differentially expressed genes in targeted therapy of colon cancer.

doi: 10.1038/s41598-025-00011-8

Figure Lengend Snippet: Fig. 6. Correlation analysis of ZG16 and CLCA1 expression. The Correlation Analysis of ZG16 and CLCA1 Expression in TCGA and GSE39582 dataset.

Article Snippet: The membranes were closed in 5% defatted milk for 2 h at room temperature and incubated with primary antibodies CLCA1 (Abconlal, CAT#A15041,1:10,000), ZG16 (Proteintech, CAT#17397-1-AP,1:5000), and β-ACTIN (Servicebio, CAT#GB11001,1:2000) overnight at 4 °C, followed by incubation with the secondary antibodies for 1 h. Finally, the indicated proteins were visualized using West Pico chemiluminescent substrate (Thermo Fisher Scientific, USA).

Techniques: Expressing

Journal: Scientific reports

Article Title: Comprehensive bioinformatics analysis was used to identify and verify differentially expressed genes in targeted therapy of colon cancer.

doi: 10.1038/s41598-025-00011-8

Figure Lengend Snippet: Fig. 7. Survival analysis. The Kaplan-Meier survival curves for overall survival (OS) and disease-free survival (DFS) in CRC patients stratified by ZG16 and CLCA1 expression levels.

Article Snippet: The membranes were closed in 5% defatted milk for 2 h at room temperature and incubated with primary antibodies CLCA1 (Abconlal, CAT#A15041,1:10,000), ZG16 (Proteintech, CAT#17397-1-AP,1:5000), and β-ACTIN (Servicebio, CAT#GB11001,1:2000) overnight at 4 °C, followed by incubation with the secondary antibodies for 1 h. Finally, the indicated proteins were visualized using West Pico chemiluminescent substrate (Thermo Fisher Scientific, USA).

Techniques: Expressing

Journal: Scientific reports

Article Title: Comprehensive bioinformatics analysis was used to identify and verify differentially expressed genes in targeted therapy of colon cancer.

doi: 10.1038/s41598-025-00011-8

Figure Lengend Snippet: Fig. 8. Overexpression of CLCA1 and ZG16 in CRC cells inhibited the proliferation, migration, and invasion of CRC cells. (A) qRT-PCR showed that CLCA1 and ZG16 were lowly expressed in CRC cells. (B) Fluorescent display virus-infected cells. (C) WB shows protein expression levels of corresponding genes in CRC cells after overexpression of two different genes. (D) The proliferative capacity of cells in the NC group and overexpressing CLCA1 and ZG16 group was determined by cck8 assay. (E) Wound healing assays were performed to detect the migration of cells in the NC group and the overexpression of the CLCA1 and ZG16 groups. (F) Transwell assay to detect the effects of overexpression of CLCA1 and ZG16 on migration and invasion of CRC cells. (G) Representative images of the colony formation assay. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: The membranes were closed in 5% defatted milk for 2 h at room temperature and incubated with primary antibodies CLCA1 (Abconlal, CAT#A15041,1:10,000), ZG16 (Proteintech, CAT#17397-1-AP,1:5000), and β-ACTIN (Servicebio, CAT#GB11001,1:2000) overnight at 4 °C, followed by incubation with the secondary antibodies for 1 h. Finally, the indicated proteins were visualized using West Pico chemiluminescent substrate (Thermo Fisher Scientific, USA).

Techniques: Over Expression, Migration, Quantitative RT-PCR, Virus, Infection, Expressing, CCK-8 Assay, Transwell Assay, Colony Assay

Journal: Scientific reports

Article Title: Comprehensive bioinformatics analysis was used to identify and verify differentially expressed genes in targeted therapy of colon cancer.

doi: 10.1038/s41598-025-00011-8

Figure Lengend Snippet: Fig. 9. Immunohistochemical analysis. Immunohistochemistry shows that CLCA1 is highly expressed in normal tissue and lowly expressed in tumor tissue, with a clear demarcation between tumor and normal tissue observed in the figure. The expression of ZG16 does not show a clear distinction between tumor and normal tissues.

Article Snippet: The membranes were closed in 5% defatted milk for 2 h at room temperature and incubated with primary antibodies CLCA1 (Abconlal, CAT#A15041,1:10,000), ZG16 (Proteintech, CAT#17397-1-AP,1:5000), and β-ACTIN (Servicebio, CAT#GB11001,1:2000) overnight at 4 °C, followed by incubation with the secondary antibodies for 1 h. Finally, the indicated proteins were visualized using West Pico chemiluminescent substrate (Thermo Fisher Scientific, USA).

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing

Journal: Scientific reports

Article Title: Comprehensive bioinformatics analysis was used to identify and verify differentially expressed genes in targeted therapy of colon cancer.

doi: 10.1038/s41598-025-00011-8

Figure Lengend Snippet: Fig. 10. Immunofluorescence double staining for ZG16 and CLCA1. The immunofluorescence staining revealed a significant difference in the expression of CLCA1 between tumor and normal tissues, whereas the expression of ZG16 did not show as clear a distinction as CLCA1.

Article Snippet: The membranes were closed in 5% defatted milk for 2 h at room temperature and incubated with primary antibodies CLCA1 (Abconlal, CAT#A15041,1:10,000), ZG16 (Proteintech, CAT#17397-1-AP,1:5000), and β-ACTIN (Servicebio, CAT#GB11001,1:2000) overnight at 4 °C, followed by incubation with the secondary antibodies for 1 h. Finally, the indicated proteins were visualized using West Pico chemiluminescent substrate (Thermo Fisher Scientific, USA).

Techniques: Immunofluorescence, Double Staining, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells

doi: 10.1016/j.jbc.2024.107432

Figure Lengend Snippet: CLCA4 self-cleavage is required to potentiate I CaCC in HEK293T cells. A , domain schematic representation of human CLCA4 constructs used in this study. The anti-CLCA1 antibody 8D3 is cross-reactive with CLCA4 and the region containing the epitope is highlighted. Domain abbreviations are as follows: CAT = MMP-like catalytic domain; SS = signal sequence; CYS = MMP-like cysteine-rich domain; VWA = von-Willebrand Type A domain; FnIII = fibronectin type-III domain; GPI = glycosylphosphatidylinositol anchor. B–D , CLCA4 is cleaved and secreted. B , Western blot (using 8D3) of cells transfected with CLCA4. Full-length CLCA4 is only detected in solubilized cells, whereas the cleaved N-terminal fragment (N-CLCA4) is found in both cell lysate and secreted into the medium. C , Western blot (using anti-FLAG M2) of cells transfected with CLCA4-FLAG. Both full-length and C-CLCA4 are only detected in solubilized cells. D , Western blot (using 8D3) of cells transfected with either WT CLCA4 or CLCA4 containing mutations to predicted active-site residues in CLCA4 MMP-like domain. These mutations (H155A and E156Q) prevent self-cleavage. E , anti-FLAG Western blot of cells transfected with CLCA-4-FLAG treated with PI-PLC to release GPI-anchored proteins. C-CLCA4-FLAG is released into media following PI-PLC treatment. F , CLCA4 self-cleavage is required to potentiate CaCC-like currents, i.e. , I CaCC . Cells were transfected with expression constructs or empty pHLsec plasmid (mock) and assayed by whole-cell patch clamp 48 h later. Representative whole-cell currents; pulse protocol on top. G , current density at +100 mV, measured at the end of the 600-ms voltage pulse. Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences between CLCA4 transfected and other conditions are indicated (Kruskal-Wallis one-way ANOVA on ranks, H (3) = 28.813, p < 0.001, followed by Dunn’s test).

Article Snippet: Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.

Techniques: Construct, Sequencing, Western Blot, Transfection, Expressing, Plasmid Preparation, Patch Clamp

Journal: The Journal of Biological Chemistry

Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells

doi: 10.1016/j.jbc.2024.107432

Figure Lengend Snippet: CLCA4-dependent I CaCC are carried by TMEM16B, and paracrine CLCA4-TMEM16B interactions are mediated by a MIDAS motif in the CLCA4 VWA domain. A , HEK293T cells transfected with scramble (siControl), TMEM16A siRNA, or TMEM16B siRNA were cultured in medium from mock-transfected cells or from cells expressing full-length CLCA4 or CLCA4 VWA domain (VWA), and assayed by whole-cell patch clamp. B and C , I CaCC were activated in CLCA4- ( B ) and VWA-conditioned ( C ) cells transfected with siControl and TMEM16A siRNA, but current density remained at background levels in cells transfected with TMEM16B siRNA. D , homology model of the CLCA4 VWA domain, based on the crystal structure of CLCA1 VWA (PDB ID: 6PYO ) . Residues composing the MIDAS site and the N-linked glycosylation site (N340) are shown as sticks. E , purified CLCA4 VWA expressed in the presence of kifunensine was treated with the deglycosylation enzyme EndoHf, resulting in a shift to lower molecular weight. F , mutation of the CLCA4 VWA glycosylation site (N340Q) results in a single band corresponding to the non-glycosylated protein. G , CLCA4-dependent I CaCC potentiation was reduced in naïve HEK293T cells conditioned with MIDAS motif double mutant S316A/T383A VWA. Experiments were performed as in ( A ). B , C and G , current density at +100 mV, measured at the end of the 600-ms voltage pulse (protocol as in Fig. 1 F ). Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences were evaluated by Kruskal-Wallis one-way ANOVA on ranks, as follows: ( B ) H (5) = 26.647, p < 0.001; ( C ) H (3) = 15.780, p = 0.001; and ( G ) H (2) = 9.267, p = 0.010. Post hoc all-pairwise comparisons between groups were determined by Dunn’s method. Statistical differences are indicated in ( B ) between mock- and CLCA4-conditioned media for each siRNA transfection in CLCA4-conditioned versus scrambled siRNA control in mock-conditioned. Statistical differences are indicated in ( C ) between mock- and CLCA4-conditioned media for each siRNA transfection. Statistical differences are indicated in ( G ) between all treatments.

Article Snippet: Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.

Techniques: Transfection, Cell Culture, Expressing, Patch Clamp, Glycoproteomics, Purification, Molecular Weight, Mutagenesis, Control

Journal: The Journal of Biological Chemistry

Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells

doi: 10.1016/j.jbc.2024.107432

Figure Lengend Snippet: Working model for CLCA family self-activation and engagement of TMEM16 family proteins. A , soluble CLCA1 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA1 and C-CLCA1 fragments. N-CLCA1 engages TMEM16A α9-α10 loop using VWA MIDAS motif. B , membrane-anchored CLCA4 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA4 and C-CLCA4 fragments. N-CLCA4 engages TMEM16B using its VWA domain.

Article Snippet: Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.

Techniques: Activation Assay, Membrane

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: Cells were fixed/permeabilized (Cat: 00-5521-00; Thermo Fisher Scientific), then incubated with the anti-CLCA1 antibody (ab180851; 1:131 in 1X Permeabilization buffer; Abcam) for 1 h in 4°C, followed by incubation with anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (1:400; Invitrogen) 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: Cells were fixed/permeabilized (Cat: 00-5521-00; Thermo Fisher Scientific), then incubated with the anti-CLCA1 antibody (ab180851; 1:131 in 1X Permeabilization buffer; Abcam) for 1 h in 4°C, followed by incubation with anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (1:400; Invitrogen) 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: Cells were fixed/permeabilized (Cat: 00-5521-00; Thermo Fisher Scientific), then incubated with the anti-CLCA1 antibody (ab180851; 1:131 in 1X Permeabilization buffer; Abcam) for 1 h in 4°C, followed by incubation with anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (1:400; Invitrogen) 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: Cells were fixed/permeabilized (Cat: 00-5521-00; Thermo Fisher Scientific), then incubated with the anti-CLCA1 antibody (ab180851; 1:131 in 1X Permeabilization buffer; Abcam) for 1 h in 4°C, followed by incubation with anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (1:400; Invitrogen) 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: Cells were fixed/permeabilized (Cat: 00-5521-00; Thermo Fisher Scientific), then incubated with the anti-CLCA1 antibody (ab180851; 1:131 in 1X Permeabilization buffer; Abcam) for 1 h in 4°C, followed by incubation with anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (1:400; Invitrogen) 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: Cells were fixed/permeabilized (Cat: 00-5521-00; Thermo Fisher Scientific), then incubated with the anti-CLCA1 antibody (ab180851; 1:131 in 1X Permeabilization buffer; Abcam) for 1 h in 4°C, followed by incubation with anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (1:400; Invitrogen) for 1 h in 4°C.

Techniques: Western Blot, Quantitative RT-PCR

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: 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