rabbit polyclonal anti chi3l1 antibody  (Proteintech)

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

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: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

Techniques: Bacteria

Journal: eLife

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

doi: 10.7554/eLife.92994

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Chi3l1 (rabbit polyclonal) , Invitrogen , PA5-95897 RRID: AB_2807699 , IHC (1:200).

Techniques: Recombinant, Plasmid Preparation, shRNA, Transfection, Construct, Purification, Bacteria, Staining, Gel Extraction, SYBR Green Assay

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A ) Immunohistochemical (IHC) staining for Chi3l1 in normal liver biopsies (Normal) and those from patients with AILI (Patient). Images shown are representative of 10 samples/group. Scale bar, 250 μm. ( B ) Enzyme-linked immunosorbent assay (ELISA) analysis of Chi3l1 in serum of healthy individuals (Normal, n = 6) and those from patients with AILI (Patient, n = 29). Data were presented as median+interquartile range. ( C, D ) Male C57B/6 mice treated with PBS or acetaminophen (APAP). ( C ) Chil1 mRNA in liver homogenates and ( D ) Chi3l1 protein levels in serum were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and ELISA at 3 and 24 hr, respectively (n = 4 mice/group). ( E, F ) Male C57B/6 (wild-typr [WT]) and Chil1 -/- mice were treated with APAP. Additionally, Chil1 -/- mice were divided into two groups treated with either PBS or recombinant mouse Chi3l1 (rmChi3l1) simultaneously with APAP (n = 4–10 mice/group). ( E ) Serum levels of ALT and ( F ) liver histology with necrotic areas outlined were evaluated 24 hr after APAP treatment. Scale bar, 250 μm. Mann-Whitney test was performed in B . Two-tailed, unpaired Student’s t-test was performed in C, D . One-way ANOVA were performed in E .

Article Snippet: Antibody , Rabbit polyclonal anti-human Chi3l1 , Proteintech , 12036–1-AP, RRID: AB_2877819 , 1:100 for IHC.

Techniques: Immunohistochemical staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Recombinant, MANN-WHITNEY, Two Tailed Test

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A ) Immunohistochemical (IHC) staining to detect platelets (CD41 + ) in healthy liver biopsies (Normal) and those from patients with AILI (Patient). Scale bar, 250 μm (n = 10/group). ( B ) Male C57B/6 mice treated with PBS or acetaminophen (APAP). Intravital microscopy analyses were performed around 3 hr post-APAP. Mɸs (cyan) and platelets (white) in liver sinusoids (red) are indicated. Representative images were chosen from intravital microscopy videos: https://bcm.box.com/s/15hmtryyrdl302mihrsm034ure87x4ea (Supplementary video 1, PBS treatment) and https://bcm.box.com/s/tuljfmstvv4lvoksx16fkxkpirkekynz (Supplementary video 2; n = 6–7 mice/group, 4–15 videos/mouse). ( C–E ) Male C57B/6 (wild-type [WT]) mice were treated with control IgG (Ctrl IgG) or an anti-CD41 antibody (α-CD41 Ab) either 3 hr before or 3 hr after APAP administration. ( C ) Serum levels of ALT and ( D ) liver histology with necrotic areas outlined were evaluated 24 hr after APAP treatment (n = 5 mice/group in C , D ). Scale bar, 250 μm. ( E ) Male C57B/6 (WT) and Chil1 -/- mice were treated with APAP. Additionally, Chil1 -/- mice were divided into two groups treated with either PBS or recombinant mouse Chi3l1 (rmChi3l1) simultaneously with APAP. Immunofluorescence (IF) staining was performed to detect intrahepatic platelets (CD41 + ) 3 hr after APAP treatment (n = 3 mice/group). Scale bar, 25 μm. Two-tailed, unpaired Student’s t-test was performed in A–C . One-way ANOVA were performed in E .

Article Snippet: Antibody , Rabbit polyclonal anti-human Chi3l1 , Proteintech , 12036–1-AP, RRID: AB_2877819 , 1:100 for IHC.

Techniques: Immunohistochemical staining, Immunohistochemistry, Intravital Microscopy, Control, Recombinant, Immunofluorescence, Staining, Two Tailed Test

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A ) Immunoprecipitation with anti-CD44 antibody was performed using liver homogenates obtained from wild-type (WT) and Cd44 -/- mice treated with acetaminophen (APAP) for 2 hr. Input proteins and immune-precipitated proteins were blotted with the indicated antibodies. ( B ) Interferometry measurement of the binding kinetics of human His-Chi3l1 with human Fc-CD44. ( C ) His-tagged control GFP and human Chi3l1 were incubated with recombinant human CD44. Proteins bound to Chi3l1 were immune-precipitated with an anti-His antibody. Input proteins and immune-precipitated proteins were blotted with indicated antibodies. ( D–F ) Male WT mice were treated with APAP and Cd44 -/- mice were treated with PBS or recombinant mouse Chi3l1 (rmChi3l1) plus APAP. ( D ) Serum levels of ALT and ( E ) liver histology with necrotic areas outlined were evaluated 24 hr after APAP treatment (n = 4–9 mice/group in A , B ). Scale bar, 250 μm. ( F ) Immunofluorescence (IF) staining was performed to detect intrahepatic platelets (CD41 + ) 3 hr after APAP treatment (n = 3 mice/group). Scale bar, 25 μm. One-way ANOVA were performed in D .

Article Snippet: Antibody , Rabbit polyclonal anti-human Chi3l1 , Proteintech , 12036–1-AP, RRID: AB_2877819 , 1:100 for IHC.

Techniques: Immunoprecipitation, Binding Assay, Control, Incubation, Recombinant, Immunofluorescence, Staining

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A–C ) Chil1 -/- mice reconstituted with recombinant mouse Chi3l1 (rmChi3l1) were treated with either Ctrl IgG or α-CD44 Ab 30 min prior to acetaminophen (APAP) challenge. ( A ) Serum levels of ALT and ( B ) liver histology with necrotic areas outlined were evaluated 24 hr after APAP treatment (n = 4–5 mice/group). Scale bar, 250 μm. ( C ) Immunofluorescence (IF) staining was performed to detect intrahepatic platelets (CD41 + ) 3 hr after APAP treatment (n = 3 mice/group). Scale bar, 25 μm. ( D ) Flow cytometry analysis was performed to identify Chi3l1-binding cells among liver non-parenchymal cells (NPCs) isolated from wild-type (WT) mice treated with APAP for 2 hr. CD44 + cells were gated from single live cells. CD44 + cells that bind to rmChi3l1 were further gated. The Chi3l1 + CD44 + cells were then identified by markers for various cell types, including CD45 + CD146 - F4/80 + (Mɸs), CD45 - CD146 + (liver sinusoidal endothelial cells [LSECs]), and Ly6G + (neutrophils). Two-tailed, unpaired Student’s t-test was performed in A .

Article Snippet: Antibody , Rabbit polyclonal anti-human Chi3l1 , Proteintech , 12036–1-AP, RRID: AB_2877819 , 1:100 for IHC.

Techniques: Recombinant, Immunofluorescence, Staining, Flow Cytometry, Binding Assay, Isolation, Two Tailed Test

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: Male wild-type (WT), Chil1 -/- , Cd44 -/- mice were treated with APAP (n = 3 mice/group). ( A ) glutathione (GSH) levels in the liver were measured at indicated time points by HPLC. ( B ) Hepatic protein levels of cytochrome P450 2E1 (CYP2E1) were measured by Western blotting after mice were fasted overnight without APAP treatment. ( C ) N -acetyl- p -benzoquinone imine (NAPQI)-protein adducts in liver were measured by Western blotting 2 hr after APAP treatment.

Article Snippet: Antibody , Rabbit polyclonal anti-human Chi3l1 , Proteintech , 12036–1-AP, RRID: AB_2877819 , 1:100 for IHC.

Techniques: Western Blot

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: Female WT, Chil1 -/- and Cd44 -/- mice were treated with acetaminophen (APAP). Serum ALT levels were measured at 6 and 24 hr after APAP treatment (n = 6–8 mice/group). One-way ANOVA was performed.

Article Snippet: Antibody , Rabbit polyclonal anti-human Chi3l1 , Proteintech , 12036–1-AP, RRID: AB_2877819 , 1:100 for IHC.

Techniques:

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A ) Male WT, Chil1 -/- , Cd44 -/- mice were treated with acetaminophen (APAP) (n = 4 mice/group). After 3 hr, mice were sacrificed and Mɸs were isolated to measure mRNA levels of various adhesion molecules, including selectin P ligand ( Selplg ), Cd40 , melanoma cell adhesion molecule ( Mcam ), Fc receptor ( Fcr ), intercellular adhesion molecule 1 ( Icam1 ), lymphocyte function-associated antigen 1 ( Lfa1 ), von Willebrand factor ( Vwf ), and podoplanin ( Pdpn ). ( B, C ) Wild-type (WT) mice were treated with APAP. Chil1 -/- and Cd44 -/- mice were treated with PBS or rmChi3l1 followed by APAP challenge simultaneously and mice were sacrificed 3 hr after APAP (n = 3 mice/group). ( B ) Mɸs were isolated and mRNA levels of Pdpn in Mɸs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). ( C ) Immunofluorescence (IF) staining of liver sections for podoplanin and F4/80 is shown and the proportions of Mɸs that express Pdpn were quantified, Scale bar, 25 μm. ( D–F ) Chil1 -/- mice reconstituted with rmChi3l1 were treated with either Ctrl IgG or α-podoplanin Ab for 16 hr and subsequently challenged with APAP. ( D ) Serum levels of ALT and ( E ) liver histology were evaluated 24 hr after APAP treatment (n = 6 mice/group). Scale bar, 250 μm. ( F ) IF staining for intrahepatic platelets (CD41 + ) and Mɸs (F4/80+) was performed 3 hr after APAP (n = 3 mice/group). Scale bar, 25 μm. One-way ANOVA were performed in A–C . Two-tailed, unpaired Student’s t-test was performed in D .

Article Snippet: Antibody , Rabbit polyclonal anti-human Chi3l1 , Proteintech , 12036–1-AP, RRID: AB_2877819 , 1:100 for IHC.

Techniques: Isolation, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Staining, Two Tailed Test

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A–C ) Male C57B/6 mice were treated with acetaminophen (APAP) for 3 hr, followed by intraperitoneally ( i.p. ) injection of either a control IgG (Ctrl IgG) or an anti-mouse Chi3l1 Ab (α-mChi3l1 Ab, C59). ( A ) Immunofluorescence (IF) staining for intrahepatic platelets (CD41 + ) was performed 6 hr after APAP treatment (n = 3 mice/group). Scale bar, 25 μm. ( B ) Serum levels of ALT and ( C ) liver histology were evaluated 24 hr after APAP treatment (n = 4–6 mice/group). Scale bar, 250 μm. ( D–F ) Chil1 -/- mice were treated with APAP plus PBS or recombinant human Chi3l1 (rhChi3l1) for 3 hr as indicated and APAP plus rhChi3l1 treatment group were either without treatment or treated with a control IgG (Ctrl IgG) or an anti-human Chi3l1 Ab (α-hChi3l1 Ab, C7). ( D ) IF staining was performed to identify intrahepatic platelets (CD41 + ) 6 hr after APAP treatment. Scale bar, 25 μm. ( E ) Serum levels of ALT and ( F ) liver histology were evaluated 24 hr after APAP treatment. Scale bar, 250 μm (n = 5–10 mice/group in D–F ). Two-tailed, unpaired Student’s t-test was performed in B . One-way ANOVA were performed in E .

Article Snippet: Antibody , Rabbit polyclonal anti-human Chi3l1 , Proteintech , 12036–1-AP, RRID: AB_2877819 , 1:100 for IHC.

Techniques: Injection, Control, Immunofluorescence, Staining, Recombinant, Two Tailed Test

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: Acetaminophen (APAP) overdose induces chitinase 3-like-1 (Chi3l1) expression, which binds CD44 on Mɸs and promotes Mɸs-mediated platelets recruitment through podoplanin/Clec-2 (C-type lectin-like receptor 2) interaction. Recruited platelets further contribute to APAP-induced liver injury (AILI).

Article Snippet: Antibody , Rabbit polyclonal anti-human Chi3l1 , Proteintech , 12036–1-AP, RRID: AB_2877819 , 1:100 for IHC.

Techniques: Expressing

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-human Chi3l1 , Proteintech , 12036–1-AP, RRID: AB_2877819 , 1:100 for IHC.

Techniques: Recombinant, Diagnostic Assay, Intravital Microscopy

Journal: The FASEB Journal

Article Title: Inflammatory‐sensitive CHI3L1 protects nucleus pulposus via AKT3 signaling during intervertebral disc degeneration

doi: 10.1096/fj.201902096r

Figure Lengend Snippet: FIGURE 1 CHI3L1 expression is significantly elevated in degenerated NP tissue. A, The GO function classification analysis of high- throughput label-free proteomics detecting differentially expressed proteins in IDD. The chart shows the number of differentially expressed proteins that are categorized in each GO term of the three GO categories as biological processes, cellular components, and molecular functions. The listed GO terms have passed the significance examine (P < .05). The red bar represents upregulated proteins in IDD in each GO term, whereas the green bar represents the downregulated proteins. B, The top 20 enriched GO terms that are significant in IDD NP samples. The bubble area represents the amount of protein, and the bubble color represents the P value. C, Six-way Venn diagram shows the shared upregulated differentially expressed proteins in IDD among the six GO terms that are the top two of each GO category. D, qPCR detecting the mRNA level of LYZ, CHI3L1, and AEBP1 in normal (IVD) and degenerated NP (IDD) tissues, n = 5. E, Representative images of hematoxylin-eosin staining (HE), Masson staining, Safranin O-fast green (SOFG) staining, and immunohistochemical staining of CHI3L1 expression in normal (IVD) and degenerated (IDD) human NP tissues. Quantifications of positive cells in the immunohistochemical analysis were shown in the right panels. Data are shown as mean ± SD. *P < .05; **P < .01

Article Snippet: Next, the sections were incubated at 4°C overnight with the following primary antibodies: rabbit polyclonal antibody against CHI3L1 (1:100 dilution), rabbit polyclonal antibody against AKT3 (1:100 dilution), and rabbit polyclonal antibody against OCN (1:100 dilution) (Proteintech).

Techniques: Expressing, High Throughput Screening Assay, Staining, Immunohistochemical staining

Journal: The FASEB Journal

Article Title: Inflammatory‐sensitive CHI3L1 protects nucleus pulposus via AKT3 signaling during intervertebral disc degeneration

doi: 10.1096/fj.201902096r

Figure Lengend Snippet: FIGURE 2 CHI3L1 is inflammatory sensitive and located mainly in NP tissue. A, Representative histological images of mouse intervertebral discs in HE, SOFG staining and immunohistochemical staining of CHI3L1 expression at various age point. Degenerated disc samples are collected from needle punctured IDD model mice. n = 5. Quantifications of positive cells in the immunohistochemical analysis were shown in (B). C, qPCR detection of CHI3L1, ACAN, CHSY1, and ADAMTS4 mRNA expression in human IDD and IVD tissue sample derived primary NP cells, n = 5. D, The expression of CHI3L1 during inflammation-induced in vitro NP cell degeneration is tested using qPCR and Western Blot. IL-1β 25 μg/mL or TNF-α 50 μg/mL are treated for 48 hours before detection. E, ELISA assay is used to detect the expression of CHI3L1 in NP cell supernatant after addition of IL-1β or TNF-α, n = 3. All experiments are repeated at least three times, and GAPDH is used as an internal control. Data are shown as mean ± SD. *P < .05; **P < .01

Article Snippet: Next, the sections were incubated at 4°C overnight with the following primary antibodies: rabbit polyclonal antibody against CHI3L1 (1:100 dilution), rabbit polyclonal antibody against AKT3 (1:100 dilution), and rabbit polyclonal antibody against OCN (1:100 dilution) (Proteintech).

Techniques: Staining, Immunohistochemical staining, Expressing, Derivative Assay, In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: The FASEB Journal

Article Title: Inflammatory‐sensitive CHI3L1 protects nucleus pulposus via AKT3 signaling during intervertebral disc degeneration

doi: 10.1096/fj.201902096r

Figure Lengend Snippet: FIGURE 3 CHI3L1 shows protective effects against inflammatory agitation. A-C, qPCR analysis showing the relative mRNA level of ACAN, CHSY1, and COL2 expression under each treatment groups. D-F, qPCR analysis showing the relative mRNA level of MMP1, MMP3, MMP13, ADAMTS4, and ADAMTS5 expression under each treatment groups. G, Flow chart of Matrigel-coated transwell assay. H, The microscopy evaluation of cell migration in different groups. Relative migrated cell level was evaluated and shown in the right panel. NC was used as a control. All experiments were repeated at least three times, and GAPDH is used as an internal control. Data are shown as mean ± SD. *P < .05; **P < .01

Article Snippet: Next, the sections were incubated at 4°C overnight with the following primary antibodies: rabbit polyclonal antibody against CHI3L1 (1:100 dilution), rabbit polyclonal antibody against AKT3 (1:100 dilution), and rabbit polyclonal antibody against OCN (1:100 dilution) (Proteintech).

Techniques: Expressing, Transwell Assay, Microscopy, Migration, Control

Journal: The FASEB Journal

Article Title: Inflammatory‐sensitive CHI3L1 protects nucleus pulposus via AKT3 signaling during intervertebral disc degeneration

doi: 10.1096/fj.201902096r

Figure Lengend Snippet: FIGURE 4 High-throughput RNA sequencing showing AKT3 is a main target of CHI3L1. A, RNA-seq was used to detect the differential expressed genes of CHI3L1 overexpressed degenerated NP cells induced by IL-1β or TNF-α (see also Figure S2A), the heatmap of unsupervised hierarchical cluster analysis showing the differentially expressed genes in IL-1β dataset. Each row represents the relative expression level of each gene in different samples. B, The Pearson coefficient between each sample based on the sequencing results. The greater the color depth, the closer R2 approaches 1, the stronger the correlation between the two groups. C, The volcano plot showing the significantly differentially expressed genes between CHI3L1 overexpressed and normal degenerated NP cells induced by IL-1β. D, The KEGG pathway enrichment analysis showing the most abundant GO terms in the IL-1β dataset, the bubble area represented the number of genes enriched in each term, and the color represents the significance. E-F, qPCR analysis showing the relative mRNA expression levels of the candidate CHI3L1 target gene (E) or related pathway gene (F). The qPCR experiments were repeated three times, and GAPDH is used as an internal control. Data are shown as mean ± SD. *P < .05; **P < .01

Article Snippet: Next, the sections were incubated at 4°C overnight with the following primary antibodies: rabbit polyclonal antibody against CHI3L1 (1:100 dilution), rabbit polyclonal antibody against AKT3 (1:100 dilution), and rabbit polyclonal antibody against OCN (1:100 dilution) (Proteintech).

Techniques: High Throughput Screening Assay, RNA Sequencing, Expressing, Sequencing, Control

Journal: The FASEB Journal

Article Title: Inflammatory‐sensitive CHI3L1 protects nucleus pulposus via AKT3 signaling during intervertebral disc degeneration

doi: 10.1096/fj.201902096r

Figure Lengend Snippet: FIGURE 5 CHI3L1 attenuates NP cell degeneration via AKT3 signaling. A-C, The mRNA levels of ACAN, CHSY1 and COL2 expression in degenerated NP cell (induced by IL-1β) were measured by qPCR analysis. GSK2141795 is an AKT3-sensitive inhibitor, and SC79 is an AKT activator. D-F, The mRNA levels of MMP1, MMP3, MMP13, ADAMTS4, and ADAMTS5 expression in degenerated NP cell (induced by IL-1β) were measured by qPCR analysis. G, The protein levels of MMP1, MMP3, MMP13, ADAMTS4, ACAN, CHSY1, and GAPDH expression were assessed by Western Blot. H, Matrigel-coated transwell assay evaluating the cell migration in different groups. Relative quantitative cell level was evaluated by microscopy and shown in the right panel. NC was used as a control. All experiments were repeated at least three times. and GAPDH is used as an internal control in qPCR analysis. Data are shown as mean ± SD. *P < .05; **P < .01

Article Snippet: Next, the sections were incubated at 4°C overnight with the following primary antibodies: rabbit polyclonal antibody against CHI3L1 (1:100 dilution), rabbit polyclonal antibody against AKT3 (1:100 dilution), and rabbit polyclonal antibody against OCN (1:100 dilution) (Proteintech).

Techniques: Expressing, Western Blot, Transwell Assay, Migration, Microscopy, Control

Journal: The FASEB Journal

Article Title: Inflammatory‐sensitive CHI3L1 protects nucleus pulposus via AKT3 signaling during intervertebral disc degeneration

doi: 10.1096/fj.201902096r

Figure Lengend Snippet: FIGURE 6 CHI3L1 autocrine is vital to the survival and anti-degeneration effect of NP cells. A, qPCR analysis showing the mRNA levels of MMP13, P21, and CCND1 expression after different doses of recombinant CHI3L1 treatment (YKL-40) in degenerated NP cell induced by IL-1β. B, The Western Blot analysis showing the protein level changes of AKT3, p-AKT3 (phospho S472), GSK3b, p-GSK3b (phospho Y216), MMP13, and GAPDH expression according to (A). C, The mRNA levels of MMP13, P21 and CCND1 expression were measured by qPCR analysis after different doses of polyclonal anti-CHI3L1 antibody treatment in degenerated NP cell induced by IL-1β. D, The protein levels of AKT3, p-AKT3 (phospho S472), GSK3b, p-GSK3b (phospho Y216), MMP13, and GAPDH expression were measured by Western Blot analysis. E-F, The qPCR analysis detecting the mRNA expression of degeneration-related genes under different doses of recombinant CHI3L1 (E) and polyclonal anti- CHI3L1 antibody treatment (F). Annexin V-FITC/PI Double-staining apoptosis assay showing the percentage of apoptotic cell percentage of different doses of recombinant CHI3L1 (G) and polyclonal anti-CHI3L1 antibody (H) treated degenerated NP cell induced by IL-1β. The effect of AKT3 activation (SC79) and inhibition (GSK2141795) on NP cell apoptosis is also tested using Annexin V-FITC/PI Double-staining apoptosis assay (I) combined with recombinant CHI3L1 treatment (rcCHI3L1) and CHI3L1 knockdown using siRNA (siCHI3L1). The mean percentage of early apoptotic cells and late apoptotic cells in each group were shown in the left panel (G-I). All experiments were repeated three times, and GAPDH is used as an internal control. Data are shown as mean ± SD. *P < .05; **P < .01

Article Snippet: Next, the sections were incubated at 4°C overnight with the following primary antibodies: rabbit polyclonal antibody against CHI3L1 (1:100 dilution), rabbit polyclonal antibody against AKT3 (1:100 dilution), and rabbit polyclonal antibody against OCN (1:100 dilution) (Proteintech).

Techniques: Expressing, Recombinant, Western Blot, Double Staining, Apoptosis Assay, Activation Assay, Inhibition, Knockdown, Control

Journal: The FASEB Journal

Article Title: Inflammatory‐sensitive CHI3L1 protects nucleus pulposus via AKT3 signaling during intervertebral disc degeneration

doi: 10.1096/fj.201902096r

Figure Lengend Snippet: FIGURE 7 CHI3L1-AKT3 axis is functional in both human and mice IDD. A, Representative immunohistochemistry images of intervertebral disc tissues showing the expression of CHI3L1, AKT3 and p-AKT3 (phospho S472) expression. The degenerative grade was classified using the Pfirrmann grading system. Five samples from different individual was analyzed in each group. B, Quantitative analysis of the proportion of CHI3L1-, AKT3-, and p-AKT3-positive cells in the immunohistochemistry staining in (A). NC was used as a control. C, Summery on the mechanism of CHI3L1 in intervertebral disc degeneration. Data are shown as mean ± SD. *P < .05, **P < .01

Article Snippet: Next, the sections were incubated at 4°C overnight with the following primary antibodies: rabbit polyclonal antibody against CHI3L1 (1:100 dilution), rabbit polyclonal antibody against AKT3 (1:100 dilution), and rabbit polyclonal antibody against OCN (1:100 dilution) (Proteintech).

Techniques: Functional Assay, Immunohistochemistry, Expressing, Staining, Control