sh trc  (Addgene inc)

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A ) Immunohistochemical (IHC) staining to detect Chi3l1 in both ileum and colon from germ-free and wildtype mice. Ctrl (wildtype mice without application of first antibody), WT (wildtype C57B/6J mice). Red arrows indicate Chi3l1-expressing cells. Scale bars, 50 μm (Ctrl, Germ-free, WT). The number of Chi3l1-positive cells in each field of view (FOV) was analyzed. ( B ) Ileum and colon were collected from wildtype mice and stained with ChgA (green), Chi3l1(red), and nuclear DAPI (blue) in ileum and UEA-1 (green), Chi3l1 (red), and nuclear DAPI (blue) in colon. Scale bars, 50 μm. Ctrl (without application of first antibody), WT (wildtype C57BL/6J mice). ( C ) Western blot to detect Chi3l1 protein expression in DLD-1 cells after bacteria mix infection for 12 hr. Bacteria mix are total bacteria extracted from feces of wildtype mice. ( D ) Western blot to detect Chi3l1 protein expression in DLD-1 cells after Staphylococcus sciuri and E. coli infection for 12 hr. Staphylococcus sciuri and E. coli are isolated from bacteria mix and verified by 16S rRNA sequencing. Three independent experimental results are showed. ( E ) Western blot to detect Chi3l1 protein expression in DLD-1 cells after treatment with heat-killed E. coli for 12 hr. Three independent experimental results are showed. ( F ) Western blot to detect Chi3l1 protein expression in DLD-1 cells after treatment with 100 pg/mL lipopolysaccharides (LPS) for 12 hr. Three independent experimental results are showed. ( G ) Immunofluorescence to detect Chi3l1 protein expression in DLD-1 cells after treatment with 100 pg/mL LPS for 12 hr. Scale bars, 20 μm. The presence of cells in the untreated sample is annotated using white dashed lines based on the overexposure. All data above represent at least three independent experiments. Representative images are shown in ( A, B ), n = 3 mice/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant difference, error bar indicates SEM. Figure 1—source data 1. File containing original western blots for , indicating the relevant bands. Figure 1—source data 2. Original files for western blot analysis displayed in . Figure 1—source data 3. Numerical data of .

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Staining, Western Blot, Bacteria, Infection, Isolation, Sequencing, Immunofluorescence

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A ) Ileum and colon were collected from Chil1 -EGFP reporter mice and stained with DCLK1 (red), Chi3l1 (green), and nuclear DAPI (blue). Scale bars, 20 μm. Representative images are shown, n = 3 mice/group. ( B ) The construction, genotyping strategy, and genotyping results of Chil1 -EGFP reporter mice. P: positive control; Wt: wildtype control; Neg: Blank control (ddH 2 O); M: DNA Ladder. Figure 1—figure supplement 1—source data 1. File containing original DNA gels for , indicating the relevant bands. Figure 1—figure supplement 1—source data 2. Original files for DNA gels for .

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques: Staining, Positive Control, Control

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A ) Structural comparison between chitin and PGN. Both chitin and PGN contain N-acetylglucosamine (GlcNAc) and have β–1,4-glycosidic bonds in their structures. However, chitin is purely a polysaccharide, while PGN includes a peptide component that forms cross-links between chains . ( B ) Gram-positive bacteria ( E. faecalis , S. saprophyticus ) and Gram-negative bacteria ( E. coli ) were incubated with 1 μg of recombinant mouse Chi3l1 protein (rmChi3l1), respectively. Proteins bound to indicated bacteria were precipitated by centrifugation. Western blot was used to detect rmChi3l1 in Pellet, Supernatant (unbound proteins) and Last Wash (last wash unbound proteins). ( C ) Insoluble PGN were incubated with either recombinant mouse Chi3l1 protein (rmChi3l1) or bovine serum albumin (BSA). Proteins bound to PGN were precipitated by centrifugation. Silver staining was used to detect rmChi3l1 in Input, Supernatant (unbound proteins), Pellet and Last Wash (last wash unbound proteins). ( D ) Insoluble PGN were incubated with recombinant human Chi3l1 protein (rhChi3l1). Proteins bound to PGN were precipitated by centrifugation. Silver staining was used to detect rhChi3l1 in Input, Supernatant (unbound proteins), Pellet and Last Wash (last wash unbound proteins). All data above represent at least three independent experiments. ( E ) Insoluble PGN or chitin was incubated with rmChi3l1. Chi3l1 bound to PGN (upper panel) and chitin (lower panel) was precipitated and detected by silver staining. The supernatant represents the last wash, and the pellet contains proteins precipitated by either PGN or chitin. ( F ) Relative DLD-1 bacterial binding preference after treatment with K12 or GlmM, a PGN synthesis-deficient mutant. Colony-forming units (CFU) were counted, and GlmM CFU were normalized to 1. ( G ) Relative K12 bacterial adhesion preference after DLD-1 cells were transfected without (Mock), or with scramble shRNA (shCK), or with sh Chil1 . CFU were counted, and the Mock group were normalized to 1. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant difference, error bar indicates SEM. Figure 2—source data 1. File containing original western blots for and silver staining for , indicating the relevant bands. Figure 2—source data 2. Original files for western blot analysis displayed in and silver staining for . Figure 2—source data 3. Numerical data of .

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques: Comparison, Bacteria, Incubation, Recombinant, Centrifugation, Western Blot, Silver Staining, Binding Assay, Mutagenesis, Transfection, shRNA

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A ) The construction, genotyping strategy and genotyping results of Chil1 -/- mice. P: positive control; Wt: wildtype control; Neg: Blank control(ddH2O); M: DNA Ladder. ( B ) The construction, genotyping strategy and genotyping results of IEC ∆ Chil1 mice. P: positive control; Wt: wildtype control; Neg: Blank control (ddH 2 O); M: DNA Ladder. PCR① and ② implicated flox, ③implicated cre. Figure 3—figure supplement 1—source data 1. File containing original DNA gels for , indicating the relevant bands. Figure 3—figure supplement 1—source data 2. Original files for DNA gels for .

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques: Positive Control, Control

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A, C, D, G ) Female Villin-cre and IEC ∆ Chil1 littermates continue to cage together after weaning for 8 weeks. Microbial communities in feces and intestinal lumen were characterized by 16S rRNA sequencing. n = 7 or 10/group. ( A ) Alpha diversity analysis of colon contents between Villin-cre and IEC ∆ Chil1 littermates. ( B ) qPCR analysis of total bacteria in the feces and ileum, colon luminal microbial communities of Villin-cre and IEC ∆ Chil1 littermates. Values for each bacterial group are expressed relative to total 16S rRNA levels. n = 5–10/group. ( C ) Principal component analysis of weighed UniFrac distances of 16S community profiles of Villin-cre and IEC ∆ Chil1 littermates feces (binary-jaccard). ( D ) Relative abundance of Gram-positive and Gram-negative bacteria in colon contents of Villin-cre and IEC ∆ Chil1 littermates are shown. ( E ) Lipoteichoic acid (LTA) (green) was detected by immunofluorescence in colon sections of Villin-cre and IEC ∆ Chil1 littermates. Nuclei were detected with DAPI. Scale bars, 50 μm. The average fluorescence intensity in each field of view (FOV) was analyzed. ( F ) Fluorescence in situ hybridization (FISH) detection of Gram-positive bacteria (red) in the colon of Villin-cre and IEC ∆ Chil1 littermates, nuclei were detected with DAPI (blue). Scale bars, 50 μm. The average fluorescence intensity in each FOV was analyzed. ( G ) Relative abundance of Gram-positive bacteria genera in colon lumen of Villin-cre and IEC ∆ Chil1 littermates. ( H ) Female wildtype and Chil1 -/- littermates continue to cage together after weaning for 8 weeks. Microbial communities in feces were characterized by 16S rRNA sequencing. n = 3 mice/group. Representative images are shown in ( E, F ), n = 4–5/3-6 mice/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant difference, error bar indicates SEM. Figure 3—source data 1. Numerical data of .

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques: Sequencing, Bacteria, Immunofluorescence, Fluorescence, In Situ Hybridization

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A ) qPCR analysis of Turicibacter in the feces of Villin-cre and IEC ∆ Chil1 is shown. Values for each bacterial group are expressed relative to total 16S rRNA levels. p-Vaule is indicated, error bar indicates SEM. Figure 3—figure supplement 2—source data 1. Numerical data of .

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques:

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A ) Immunohistochemical (IHC) staining to detect Chi3l1 in colon mucus layer from wildtype mice. Ctrl (without application of ant-Chi3l1 antibody), WT (wildtype C57BL/6J mice). Black dotted line outlines mucus layer. Scale bars, 50 μm (Ctrl, WT). ( B ) Colons were collected from wildtype mice and stained with UEA-1 (green), Chi3l1 (red), and nuclear DAPI (blue). Ctrl (without application of first antibody), WT (wildtype C57BL/6J mice). Scale bars, 50 μm (Ctrl, WT). ( C ) Stool, ileum, and colon tissues were collected from wildtype mice. Western blot was used to detect Chi3l1 expression in these samples. n = 3 mice/sample. ( D ) Both luminal and mucus-associated proteins of either ileum or colon were extracted. Western blot was used to detect Chi3l1 expression in these samples. lumen (luminal proteins), and mucus (mucus-associated proteins). n = 3 mice/sample. ( E, F ) qPCR analysis of specific bacteria in the ileum and colon mucus microbial communities of wildtype and Chil1 -/- littermates. ( E ) qPCR analysis of Gram-positive bacteria is shown. ( F ) qPCR analysis of Gram-positive bacteria is shown. Values for each bacterial group are expressed relative to total 16S rRNA levels. WT (wildtype C57BL/6J mice). n = 6–8/group. ( G ) Rectal injection of both wildtype and Chil1 -/- mice with FDAA-labeled E. faecalis (a Gram-positive bacteria strain) for 4 hr. Colon sections were collected and colonization of E. faecalis was examined under microscope. Nuclei were stained with DAPI. Scale bars, 50 μm (WT, Chil1 -/- ). Representative images are shown in ( A, B, G ), n = 3 mice/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant difference, error bar indicates SEM. Figure 4—source data 1. File containing original western blots for , indicating the relevant bands. Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. Numerical data of .

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques: Immunohistochemical staining, Immunohistochemistry, Staining, Western Blot, Expressing, Bacteria, Injection, Labeling, Microscopy

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A ) Rectal injection of both wildtype and Chil1 -/- mice with mCherry-OP50 (a strain of E. coli expressing mCherry) for 4 hr. Colon sections were collected and colonization of OP50 was examined under microscope. Nuclei were stained with DAPI. n = 3–4 mice/group. The average fluorescence intensity in each field of view (FOV) was analyzed. ( B ) Periodic acid–Schiff and Alcian blue (AB-PAS) staining in the colons of WT and Chil1 -/- littermates. Scale bars, 100 μm. The mean width of mucus layer in each FOV was analyzed. ( C ) Immunofluorescence staining to detect Mucin 2 (green) and nuclear DAPI (blue) in colon from WT and Chil1 -/- littermates. Scale bars, 50 μm. Representative images are shown in ( C, D ), n = 4 mice/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant difference, error bar indicates SEM. Figure 4—figure supplement 1—source data 1. Numerical data of .

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques: Injection, Expressing, Microscopy, Staining, Fluorescence, Immunofluorescence

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A ) Chil1 mRNA relative expression in colon tissues of patients without gut disease (controls, n = 35) or with Crohn’s disease (CD, n = 40), ulcerative colitis (UC, n = 40) (GEO datasets: SRP303290). ( B ) Schematic model of the experimental design. Both Villin-cre and IEC ∆ Chil1 littermates were fed with 2% dextran sodium sulfate (DSS) in drinking water to induce colitis. ( C ) Weight change of Villin-cre and IEC ∆ Chil1 littermates during DSS feeding. Weight change (%) = Current weight/Initial weight. ( D ) Representative colonic length from Normal and DSS-treated Villin-cre and IEC ∆ Chil1 littermates (left) and the statistics of colonic length (right). ( E ) H&E staining of mice colon from Normal and DSS-treated Villin-cre and IEC ∆ Chil1 littermates. The inflamed areas are outlined by white dotted line, scale bars = 100 μm. ( F ) Schematic of the experimental design. First, antibiotics were used to eliminate gut microbiota for 10 days, and then either fecal microbiota from Villin-cre mice (FMT) or Lactobacillus reuteri were transplanted back to IEC ∆ Chil1 mice orally every day for 2 weeks. Finally, colitis mouse model was constructed by 2% DSS feeding in drinking water for another 7 days. ( G–I ) Villin-cre and IEC ∆ Chil1 were only fed with 2% DSS in drinking water for 7 days. IEC ∆ Chil1 + FMT(Villin-cre), and IEC ∆ Chil1 + Lactobacillus were constructed as described in ( F ). ( G ) Weight change of Villin-cre, IEC ∆ Chil1 , IEC ∆ Chil1 + FMT(Villin-cre), and IEC ∆ Chil1 + Lactobacillus mice during DSS feeding. ( H ) Representative colonic length from Villin-cre, IEC ∆ Chil1 , IEC ∆ Chil1 + FMT(Villin-cre), and IEC ∆ Chil1 + Lactobacillus mice (left) and the statistics of colonic length (right). n = 3–6/group. ( I ) H&E staining of mice colon from Villin-cre, IEC ∆ Chil1 , IEC ∆ Chil1 + FMT(Villin-cre), and IEC ∆ Chil1 + Lactobacillus mice after DSS treatment. The inflamed area is outlined by black dotted line, scale bars = 100 μm. Representative images are shown in ( C, E, H, I ), n = 3–8 mice/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant difference, error bar indicates SEM. Figure 5—source data 1. Numerical data of .

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques: Expressing, Staining, Construct

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: ( A ) Schematic model of the experimental design. Both Villin-cre and IEC ∆ Chil1 littermates were fed with 2% dextran sodium sulfate (DSS) in drinking water to induce colitis after elimination of gut microbiota by antibiotics for 10 days. ( B ) qPCR analysis of total bacteria in the feces of Villin-cre and IEC ∆ Chil1 littermates. Values for each bacterial group are expressed relative to total 16S rRNA levels. n = 3/group. ( C ) Weight change of Villin-cre and IEC ∆ Chil1 mice during DSS feeding. ( D ) Representative colonic length from colitis Villin-cre and IEC ∆ Chil1 mice (left) and the statistics of colonic length (right). ( E ) H&E staining of colitis mice colon from Villin-cre and IEC ∆ Chil1 . The inflamed area is outlined by black dotted lines, scale bars = 100 μm. n = 4–6 mice/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significant difference, error bar indicates SEM. Figure 5—figure supplement 1—source data 1. Numerical data of .

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques: Bacteria, Staining

Journal: eLife

Article Title: Peptidoglycan-Chi3l1 interaction shapes gut microbiota in intestinal mucus layer

doi: 10.7554/eLife.92994

Figure Lengend Snippet: Intestinal epithelial cells are stimulated by the gut microbiota to express Chi3l1. Once expressed, Chi3l1 is secreted into the mucus layer where it interacts with the gut microbiota, specifically through a component of bacterial cell walls called peptidoglycan. This interaction between Chi3l1 and bacteria is beneficial for the colonization of bacteria in the mucus, particularly for Gram-positive bacteria like Lactobacillus . Moreover, a deficiency of Chi3l1 leads to an imbalance in the gut microbiota, which exacerbates colitis induced by dextran sodium sulfate (DSS).

Article Snippet: Transfected construct (human) , sh Chil1 (constructed from pLKO.1 – TRC) , This paper , Constructed from RRID: Addgene_10878 , Lentiviral construct to transfect and express the shRNA.

Techniques: Bacteria