Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a Single-cell profiling based on colonic biopsies. Left: uniform manifold approximation and projection (UMAP) plot in healthy controls (HC), uninflamed regions from patients (SC), and inflamed regions from patients (UC); middle: dot heatmap presents the gene expression in each group. Each row indicates a different cell type, while each column represents an individual gene. The color depth of each circle is correlated with the level of gene expression, and the size corresponds with the proportion of cells; right: heatmap of the top 100 genes under pseudotime condition presented in the UC group. b qRT-PCR results of relative SPINK4 level in the intestinal mucosa (CD, n = 32; UC, n = 24; N , n = 65). c Representative SPINK4 level in endoscopic specimens via Western blotting. Lines 1–2 represent healthy controls; lines 3–6 and lines 7–10 represent UC and CD patients, respectively. d , e Relative level of Spink4 in the TNBS model ( d ) at the active stage and DSS models ( e ) at the active and remission stages. f , g Differential expression of SPINK4 in TNBS-induced colitis models at the active stage ( f ) and the whole stage ( g ) via immunoblotting. Left panel: controls (lines 1–4) and TNBS administration (lines 5–8); Right panel: SPINK4 expression in injured intestines including day 2 (D2), 5 (D5), and 8 (D8) after TNBS enema. h Intestinal staining targeting SPINK4 from two models at different stages; Day 2 and Day 5 refer to the time after TNBS enema. i Double immunofluorescence staining targeting SPINK4 and MUC2 in healthy control (NC) tissue and inflammatory sections of IBD with DAPI counterstaining. Scale bar: 100 μm. j RNA localization of SPINK4 in intestinal mucosa from CD and UC patients, and negative controls (NC) showed. Red dots denote the mRNA localization of SPINK4 with hematoxylin counterstaining. Data are presented as the mean ± SEM. All tests were two-sided. Statistical significance was calculated using unpaired Student’s t test ( d , f ) and one-way analysis of variance (ANOVA) ( e , g ). Kruskal–Wallis test was performed in non-normal data ( b , c ); n = 2–4 biologically independent experiments ( c , f , g ). Source data are provided as a Source Data file.

Article Snippet: Whole organoids were harvested in cell recovery solution (Corning, USA) , followed by fixation in 10% formalin for 15 min, and permeated in 0.5% Triton-X for 20 min. After blocking in 1% BSA prepared in PBS, the organoids were incubated with antibodies against MUC2 (1:100, Genetex, USA), E-cadherin (1:100, Santa Cruz, USA), chromogranin A (CgA, 1:100, Santa Cruz), lysozyme (Lyz, 1:100, Santa Cruz) and Ki67 (1:250, Abcam, UK).

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Expressing, Staining, Double Immunofluorescence Staining, Control

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a Schematic overview of in vivo experiment; the black upwards arrow indicates rSPINK4, red indicates gefitinib, blue stands for infliximab and gray represents Pam2CSK4. The red triangles represent the pre-sensitization and enema with TNBS. The downwards arrow indicates sacrifice time. b – d Weight loss ( b ), representative colonic images ( c ) and histological scores ( d ) in the ethanol group (control, n = 8), TNBS group (TNBS + rFc, n = 8), and rSPINK4-Fc treatment group (TNBS + rSPINK4-Fc, n = 9). e Weight loss in WT H 2 O ( n = 12), KO H 2 O ( n = 8), WT mice administrated DSS solution (WT DSS, n = 8), and cKO mice administrated DSS solution (KO DSS, n = 10). f , g Appearance of colon ( f ) and intestinal length ( g ) in the four groups. h Serum level of fluorescence signal at excitation/emission wavelength of 490/530 nm. i Typical images from cKO and co-housed colitis mice presented with H&E, AB/PAS staining (arrow points to mucus layer), double immunofluorescence for MUC2 (red), CgA (pink) counterstained with DAPI dye (blue), and TEM test. Unlabeled scale bar: 100 μm. j Secretory biomarkers for GCs (MUC2), Paneth cells (Lyz), and enteroendocrine cell (CgA) in the four groups. k Representative pictures of the luminol-based in vivo endoscopic imaging (the circle represents the erosion or ulceration) in WT H 2 O ( n = 7), KO H 2 O ( n = 8), WT mice administrated DSS and rFc (WT DSS+rFc, n = 5), cKO mice administrated DSS and rFc (KO DSS+rFc, n = 7), and cKO mice with rSPINK4-Fc treatment (KO DSS+rSPINK4-Fc, n = 7). l , m Weight loss ( l ) and length changes ( m ) in the five groups. n Weight change in infliximab (IFX, n = 18), rSPINK4 ( n = 6), and combined treatment ( n = 12) compared with TNBS models (TNBS + vehicle, n = 8) and control group ( n = 8). o Representative H&E image of Swiss roll intestine. The dotted line represents the inflammatory cell infiltration, while the continuous line describes severe ulceration. Data are presented as the mean ± SEM. All tests were two-sided. Statistical significance was calculated using unpaired Student’s t test ( n ) and one-way ANOVA ( b , e , g , h , j , l , m ). Kruskal–Wallis test was performed in non-normal data ( d ); n = 3–13 biologically independent experiments ( d , h , j , l , m ). Source data are provided as a Source Data file.

Article Snippet: Whole organoids were harvested in cell recovery solution (Corning, USA) , followed by fixation in 10% formalin for 15 min, and permeated in 0.5% Triton-X for 20 min. After blocking in 1% BSA prepared in PBS, the organoids were incubated with antibodies against MUC2 (1:100, Genetex, USA), E-cadherin (1:100, Santa Cruz, USA), chromogranin A (CgA, 1:100, Santa Cruz), lysozyme (Lyz, 1:100, Santa Cruz) and Ki67 (1:250, Abcam, UK).

Techniques: In Vivo, Control, Fluorescence, Staining, Immunofluorescence, Imaging

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a Relative expression of IEC markers from different branches, including enteroendocrine cells ( Neurog3 , Insm , and Pax4 ), secretory lineage ( Muc2 , Gfi1 , Foxa2 , Reg3a , and Lyz ), M cells ( Spib ), Tuft cells ( Dclk ), stem cells ( Olfm4 , Lgr5 ), and absorptive cells ( Si ) in mouse organoids with rSPINK4 stimulation. The degree of color depth indicates the abundance of expression positively. b Levels of phosphorylated and total EGFR in NCM460 and HT-29 cells with 5 ng/mL rSPINK4 stimulation for 0–60 min. c Direct downstream molecules of the EGFR pathway detected using western blotting with rSPINK4 stimulation for 0–60 min. d Typical fluorescent images of intestinal organoids from WT and KO mice including MUC2 (green), E-cadherin (red), and DAPI (blue), with rSPINK4 (100 ng/mL) and AG1478 (10 μM) stimulation under inflammatory conditions (50 ng/mL TNF-α treatment). e Quantitative analysis of weight loss with SPINK4 and gefitinib intervention following DSS administration in the control group drinking water (control, n = 4); DSS and PBS administration group (DSS + PBS, n = 6); DSS and rSPINK4 administration group (DSS + rSPINK4, n = 5); and DSS, rSPINK4, and gefitinib administration group (DSS + rSPINK4 + gefitinib, n = 4). f Representative canonical endoscopic images with the lesion circled with white arrows. g Quantification of colonic length. h The levels of the phosphorylated and total EGFR were determined in WT and KO mouse tissues (upper panel) and rSPINK4 treatment group (lower panel). Data are presented as the mean ± SEM. All tests were two-sided. Statistical significance was calculated using unpaired Student’s t test ( h ). Besides, Kruskal–Wallis test ( h ) and one-way ANOVA ( b , e , g ) were performed for multiple comparison; n = 3–5 biologically independent experiments ( b , g , h ). Source data are provided as a Source Data file.

Article Snippet: Whole organoids were harvested in cell recovery solution (Corning, USA) , followed by fixation in 10% formalin for 15 min, and permeated in 0.5% Triton-X for 20 min. After blocking in 1% BSA prepared in PBS, the organoids were incubated with antibodies against MUC2 (1:100, Genetex, USA), E-cadherin (1:100, Santa Cruz, USA), chromogranin A (CgA, 1:100, Santa Cruz), lysozyme (Lyz, 1:100, Santa Cruz) and Ki67 (1:250, Abcam, UK).

Techniques: Expressing, Western Blot, Control, Comparison

Journal: Nature Communications

Article Title: Therapeutic potential of the secreted Kazal-type serine protease inhibitor SPINK4 in colitis

doi: 10.1038/s41467-024-50048-y

Figure Lengend Snippet: a , b SPINK4 levels in LS174T cell supernatant stimulated with Pam2CSK4 ( a ) and carbachol ( b ). c , d Relative expression of SPINK4 with 100 μg/mL Pam2CSK4 stimulation in human intestinal organoids ( c ) and with stimulation with a lower concentration (0.01 μg/mL) in mouse intestinal organoids ( d ). e SPINK4 expression presented with MUC2, Lyz, and CgA values in the intestinal organoids from WT and cKO mice with Pam2CSK4 treatment. f Gel-forming mucin and transmembrane mucin expression when SPINK4 was overexpressed or knocked out were assessed using qRT-PCR test. OE, overexpression; NC, negative control; KO, knockout. g MUC2 expression was influenced by the intrinsic SPINK4 levels in the LS174T cells and the phosphorylated EGFR levels. OE, overexpression; NC, negative control; KO, knockout. Blank indicates no vector involved. h , i The correlation between MUC2 level with SPINK4 expression in IBD. The mRNA level was normalized to β-actin level ( h ). The Pearson correlation coefficient ( r ) was used to estimate the correlation between MUC2 and SPINK4 expression ( i ). j The apparent difference in colonic permeability following Spink4 knockout (UEA1: green, EUB338: red, DAPI: blue; white arrows: the bacteria which penetrate the inner mucus layer; red triangles: the outside of the inner mucus layer. Data are presented as the mean ± SEM. All tests were two-sided. Statistical significance was calculated using unpaired Student’s t test ( d , g ) and one-way analysis of variance (ANOVA) ( a , b , g ). Mann–Whitney U test and Kruskal–Wallis test were performed on non-normal data ( c , h ); n = 3 biologically independent experiments ( a – d , g ). Source data are provided as a Source Data file.

Article Snippet: Whole organoids were harvested in cell recovery solution (Corning, USA) , followed by fixation in 10% formalin for 15 min, and permeated in 0.5% Triton-X for 20 min. After blocking in 1% BSA prepared in PBS, the organoids were incubated with antibodies against MUC2 (1:100, Genetex, USA), E-cadherin (1:100, Santa Cruz, USA), chromogranin A (CgA, 1:100, Santa Cruz), lysozyme (Lyz, 1:100, Santa Cruz) and Ki67 (1:250, Abcam, UK).

Techniques: Expressing, Concentration Assay, Quantitative RT-PCR, Over Expression, Negative Control, Knock-Out, Plasmid Preparation, Permeability, Bacteria, MANN-WHITNEY

Journal: Nature Communications

Article Title: FAM3D is essential for colon homeostasis and host defense against inflammation associated carcinogenesis

doi: 10.1038/s41467-020-19691-z

Figure Lengend Snippet: a Heatmaps of differentially expressed genes enriched in host defense responses. b mRNA expression levels of antibacterial peptides in the colons of WT or Fam3D −/− mice measured by real-time PCR. n = 8. c Reg3γ protein levels measured by immunofluorescent staining. n = 4 for each group. d GSEA analysis on Fam3D −/− vs WT EBSeq log 2 FC expression data of colonic epithelial samples. e Immunofluorescent staining for Fam3D and MUC2 in normal colon tissue. Data is representative of one experiment repeated three independent times. f Double staining of Acian blue and PAS. Yellow arrow: Acian blue positive materials; orange arrow: PAS single positive cells; orange star: dilated goblet cells, goblet cells enumerated in each crypt (10–20 crypts from each colon) and pixels of Alcian blue staining areas. Alcian blue staining areas and fluorescence intensity quantified by Image J based on six distal colon sections of four independent mice from each group. g Staining of high iron diamine/Alcian blue, dark brown, and black were sulfomucins, whereas blue staining indicates sialomucins, sulfomucin positive cells enumerated in each crypt (10–20 crypts from each colon) and the ratio of sialomucins to sulfomucins is calculated. Data is presented as the mean ± SEM. Scale bar = 50 μm. h Representative Alcian blue staining of Carnoy’s-fixed colonic sections. The thickness of the inner mucus layer (light blue band between luminal content and the mucosa) was quantified (5–10 fields from each colon) by Image J. n = 8. i Representative dual staining for UEA-I (immunofluorescence, green) and bacteria (fluorescence in situ hybridization, red), the latter using the universal EUB338 probe, on Carnoy’s-fixed colonic sections. n = 8. j PAS/Alcian blue staining of the colons from WT and Fam3D −/− mice with different ages. n = 4. Data is representative of one experiment repeated three independent times. k Quantitation of crypt length of WT and Fam3D −/− mice at different ages. Data is presented as the mean ± SEM. Statistical significance was determined by unpaired, two-tailed Student’s t test.

Article Snippet: Rat anti-EpCAM antibody (sc-53532), mouse anti-β-catenin antibody (sc-7963), rabbit anti-mouse Mucin 2 (MUC2) antibody (H300) were purchased from Santa Cruz (Dallas, TX).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Double Staining, Fluorescence, Immunofluorescence, Bacteria, In Situ Hybridization, Quantitation Assay, Two Tailed Test

Journal: Mucosal immunology

Article Title: Hypoxia-inducible factor 1 in dendritic cells is crucial for the activation of protective regulatory T cells in murine colitis.

doi: 10.1038/mi.2015.67

Figure Lengend Snippet: Figure 6 Dendritic cell-specific knockout of hypoxia-inducible factor-1a (HIF-1a) stimulates mucosal epithelium to increased production of mucins in dextran sodium sulfate (DSS) colitis. Colonic mRNA expression of (a) MUC1, (b) MUC2, and (c) MUC3 was significantly higher in DSS-treated CD11cCre/HIF-1a þ f/ þ f mice than in HIF-1a þ f/ þ f DSS-treated mice or in mice not treated with DSS. (d) Sections (4 mm) of colon tissue were stained with MUC2 (red) and 4’,6-diamidino-2-phenylindole (DAPI; blue). After treatment with DSS, the amount of colonic MUC2 protein was higher in CD11cCre/HIF- 1a þ f/ þ f mice than in HIF-1a þ f/ þ f mice (original magnification 200; scale bar ¼ 50 mm). N ¼ 5–7 per group. *Po0.05; **Po0.01; ****Po0.0001. C, control; NS, not significant.

Article Snippet: MUC2 was detected with a rabbit anti-mouse MUC2 antibody (Santa Cruz Biotechnology).

Techniques: Knock-Out, Expressing, Staining, Control

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