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anti mouse ccl2 polyclonal goat ab (R&D Systems)

Name: anti mouse ccl2 polyclonal goat ab
Brand: R&D Systems
Rating: 94 (6 reviews)

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R&D Systems anti mouse ccl2 polyclonal goat ab
Anti Mouse Ccl2 Polyclonal Goat Ab, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 6 article reviews

anti mouse ccl2 polyclonal goat ab - by Bioz Stars, 2026-02

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(A) Conditioned media (CM) was collected from primary WT and Slc4a4 KO astrocytes and subjected to LC-MS/MS-based unbiased proteomics and cytokine/chemokine array. (B) Angiogenic factors detected from LC-MS/MS-based unbiased proteomics. (C) Cytokine/chemokines that are changed in the CM from WT and Slc4a4 KO astrocytes. (D) CM from primary WT and Slc4a4 KO astrocytes cultured under either normal or oxygen-glucose deprivation (OGD) condition was collected for CCL2 measurement by ELISA. Each dot represents conditioned media collected from individual animals’ astrocytes. N = 3-5 per genotype. *p<0.05, ****p<0.01 by Student’s t-test. (E) Proposed model for the Slc4a4-CCL2-CCR2 axis regulating astrocyte-endothelia interaction. (F) Quantification of cortical CCL2 levels in the uninjured and stroked (1 dpi) brains from WT and Slc4a4-icKO mice. Data are presented as mean ± SEM. N = 3-5 mice per genotype. *p<0.05, **p<0.01 by Student’s t-test. (G) Quantification of astrocytic CCL2 expression from double immunostaining. Data are presented as mean ± SEM. n = 7-17 cells collected from N = 3-5 mice per genotype. *p<0.05 by Student’s t-test. (H) Quantification of endothelial CCR2 expression from double immunostaining. Data are presented as mean ± SEM. Each dot represents an individual animal. N = 4-5 per genotype. *p<0.05, *p<0.05 by Student’s t-test. (I) At 4 dpi, astrocytic CCL2 expression was visualized by double immunostaining of CCL2/GFAP. Endothelial and astrocytic CCR2 expression was visualized by double immunostaining of CCR2/CD31 and CCR2/Aldh1l1-GFP respectively.

Journal: bioRxiv

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway

doi: 10.1101/2023.04.03.535167

Figure Lengend Snippet: (A) Conditioned media (CM) was collected from primary WT and Slc4a4 KO astrocytes and subjected to LC-MS/MS-based unbiased proteomics and cytokine/chemokine array. (B) Angiogenic factors detected from LC-MS/MS-based unbiased proteomics. (C) Cytokine/chemokines that are changed in the CM from WT and Slc4a4 KO astrocytes. (D) CM from primary WT and Slc4a4 KO astrocytes cultured under either normal or oxygen-glucose deprivation (OGD) condition was collected for CCL2 measurement by ELISA. Each dot represents conditioned media collected from individual animals’ astrocytes. N = 3-5 per genotype. *p<0.05, ****p<0.01 by Student’s t-test. (E) Proposed model for the Slc4a4-CCL2-CCR2 axis regulating astrocyte-endothelia interaction. (F) Quantification of cortical CCL2 levels in the uninjured and stroked (1 dpi) brains from WT and Slc4a4-icKO mice. Data are presented as mean ± SEM. N = 3-5 mice per genotype. *p<0.05, **p<0.01 by Student’s t-test. (G) Quantification of astrocytic CCL2 expression from double immunostaining. Data are presented as mean ± SEM. n = 7-17 cells collected from N = 3-5 mice per genotype. *p<0.05 by Student’s t-test. (H) Quantification of endothelial CCR2 expression from double immunostaining. Data are presented as mean ± SEM. Each dot represents an individual animal. N = 4-5 per genotype. *p<0.05, *p<0.05 by Student’s t-test. (I) At 4 dpi, astrocytic CCL2 expression was visualized by double immunostaining of CCL2/GFAP. Endothelial and astrocytic CCR2 expression was visualized by double immunostaining of CCR2/CD31 and CCR2/Aldh1l1-GFP respectively.

Article Snippet: The following commercial primary antibodies were used: rabbit anti-GFAP (1:1,000; Agilent Dako), mouse anti-NeuN (1:500; Millipore), rabbit anti-GFP (1:1,000; Chromotek; PABG1-100), rabbit anti-NF1A (gift from Dr. Benjamin Deneen), rabbit anti-Sox9 (1:1,000; Millipore; AB5535), rat anti-BrdU (1:1,000; Abcam; ab6326), rabbit anti-S100b (1:1,000; Agilent Dako; Z0311), Rat anti-CD31(1:200; BD Bioscience; 55047), rabbit anti-Glut1 (1:500; Millipore; 07-1401), rabbit anti-AQP4(1:500; Sigma; A5971), rabbit anti-ZO-1 (1:250; Invitrogen;40-2200), Alexa 488 conjugated Claudin-5 (1:500; Invitrogen, 35-358-8), rabbit anti-CCL2 (1:500, PerproTech, 500-P113), goat anti-CCL2 (1:500; R&D, AF-479), ), rabbit anit-CCR2 (1:500; Abcam; ab273050), mouse anti-Cav-1 (1:500; BD Bioscience; 610406), mouse anti-pCav-1 (1:500; BD Bioscience; 611339).

Techniques: Liquid Chromatography with Mass Spectroscopy, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Double Immunostaining

(A) Mouse endothelial cells (bEnd3) were incubated with conditioned media (CM) collected from primary WT and Slc4a4 KO astrocytes with CCL2 functional blocking antibody (CCL2 ⍺FB, 15 ng/ml) or CCR2 antagonist for 24 hours. (B-D) Representative immunofluorescence images and quantification of tight junction proteins (ZO-1 and Claudin-5) of bEnd3 cells incubated with WT- or Slc4a4 KO-CM in the presence of control IgG or CCL2-FB⍺. Data are presented as mean ± SEM. Each dot represents a well collected from 3 independent experiments. *p<0.05, ***p<0.001 by two-way ANOVA. (E) Experimental setup of the transendothelial electrical resistance (TEER) assay. A monolayer of bEnd3 cells was co-cultured with WT or Slc4a4-deficient astrocytes, where astrocytes were plated at the bottom of the well and endothelial cells were plated in the insert, allowing endothelial cells to be exposed to astrocytic secreting factors without physical contact with astrocytes. CCL2 blocking antibody was added into the insert with a final concentration of 15 ng/ml. (F) The electro-resistance of the endothelial cell monolayer was measured as an indicator for the permeability of endothelial cells in the TEER assay. Data are presented as mean ± SEM. Each dot represents each independent culture. N = 4 independent assays. *p<0.05, **p<0.01 by two-way ANOVA. (G-H) Caveolin- and clathrin-mediated endothelial intracellular uptake was examined by Texas Red conjugated albumin and A488-transferrin, respectively, in bEnd3 cells incubated with WT- or Slc4a4 KO-CM with CCR2 FBα or control IgG. Data are presented as mean ± SEM. Each dot represents each independent culture. N= 3 independent assays. **p<0.01, ***p<0.001 by two-way ANOVA. (I-J) Western blot analysis of pCav-1, Cav-1 and CCR2 expression in bEnd3 cells incubated with WT- or Slc4a4 KO-CM with CCR2 FBα or control IgG. Data are presented as mean ± SEM. Each dot represents each independent culture. N = 3 independent assays. **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA. (K-L) Paracellular and transcellular endothelial transport in bEnd3 cells incubated with WT- or Slc4a4 KO-CM with CCR2 antagonist RS504393 (10 μM) or control DMSO were examined by immunostaining of ZO-1 and pCav-1. Data are presented as mean ± SEM. Each dot represents each independent culture. N = 3 independent assays. *p<0.05, **p<0.01, ***p<0.001 by two-way ANOVA. (M) A proposed mechanism by which astrocytic Slc4a4 regulates endothelial paracellular and transcellular transport pathways via the CCL2-CCR2 axis.

Journal: bioRxiv

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway

doi: 10.1101/2023.04.03.535167

Figure Lengend Snippet: (A) Mouse endothelial cells (bEnd3) were incubated with conditioned media (CM) collected from primary WT and Slc4a4 KO astrocytes with CCL2 functional blocking antibody (CCL2 ⍺FB, 15 ng/ml) or CCR2 antagonist for 24 hours. (B-D) Representative immunofluorescence images and quantification of tight junction proteins (ZO-1 and Claudin-5) of bEnd3 cells incubated with WT- or Slc4a4 KO-CM in the presence of control IgG or CCL2-FB⍺. Data are presented as mean ± SEM. Each dot represents a well collected from 3 independent experiments. *p<0.05, ***p<0.001 by two-way ANOVA. (E) Experimental setup of the transendothelial electrical resistance (TEER) assay. A monolayer of bEnd3 cells was co-cultured with WT or Slc4a4-deficient astrocytes, where astrocytes were plated at the bottom of the well and endothelial cells were plated in the insert, allowing endothelial cells to be exposed to astrocytic secreting factors without physical contact with astrocytes. CCL2 blocking antibody was added into the insert with a final concentration of 15 ng/ml. (F) The electro-resistance of the endothelial cell monolayer was measured as an indicator for the permeability of endothelial cells in the TEER assay. Data are presented as mean ± SEM. Each dot represents each independent culture. N = 4 independent assays. *p<0.05, **p<0.01 by two-way ANOVA. (G-H) Caveolin- and clathrin-mediated endothelial intracellular uptake was examined by Texas Red conjugated albumin and A488-transferrin, respectively, in bEnd3 cells incubated with WT- or Slc4a4 KO-CM with CCR2 FBα or control IgG. Data are presented as mean ± SEM. Each dot represents each independent culture. N= 3 independent assays. **p<0.01, ***p<0.001 by two-way ANOVA. (I-J) Western blot analysis of pCav-1, Cav-1 and CCR2 expression in bEnd3 cells incubated with WT- or Slc4a4 KO-CM with CCR2 FBα or control IgG. Data are presented as mean ± SEM. Each dot represents each independent culture. N = 3 independent assays. **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA. (K-L) Paracellular and transcellular endothelial transport in bEnd3 cells incubated with WT- or Slc4a4 KO-CM with CCR2 antagonist RS504393 (10 μM) or control DMSO were examined by immunostaining of ZO-1 and pCav-1. Data are presented as mean ± SEM. Each dot represents each independent culture. N = 3 independent assays. *p<0.05, **p<0.01, ***p<0.001 by two-way ANOVA. (M) A proposed mechanism by which astrocytic Slc4a4 regulates endothelial paracellular and transcellular transport pathways via the CCL2-CCR2 axis.

Techniques: Incubation, Functional Assay, Blocking Assay, Immunofluorescence, Cell Culture, Concentration Assay, Permeability, Western Blot, Expressing, Immunostaining

(A) Experimental scheme of the PTS induction in WT and Slc4a4-icKO mice, followed by intraperitoneal injection of CCL2 functional blocking antibody (CCL2-FB⍺) at 1 dpi. Brains were then harvested and analyzed at 4 dpi. (B) Representative images of protein leakage (Evans blue, fibrinogen), endothelial junctional marker expression (Claudin-5+; CD31+), endothelial pCav-1 expression and reactive astrocyte marker GFAP at the peri-lesion area in WT and Slc4a4-icKO mice with or without CCL2-FB⍺ treatment. (C) Quantification of fibrinogen intensity from immunostaining. Data are presented as mean ± SEM. Each dot represents an individual animal (N = 4-9 per group). ***p<0.001, ****p<0.0001 by two-way ANOVA. (D) Quantification of CD31 intensity from immunostaining. Data are presented as mean ± SEM. n = 2-3 blood vessels per animal and N = 4-5 animals per group. **p<0.01 ***p<0.001, ****p<0.0001 by two-way ANOVA. (E) Quantification of the intensity of Claudin-5 colocalized with CD31. Data are presented as mean ± SEM. n = 2-3 blood vessels per animal and N = 4-5 animals per group. *p<0.05, ***p<0.001 by two-way ANOVA. (F) Quantification of the intensity of pCav-1 colocalized with CD31. Data are presented as mean ± SEM. n = 4-5 blood vessels per animal and N = 4-5 animals per group. *p<0.05, ***p<0.001 by two-way ANOVA. (G) Quantification of GFAP intensity from immunostaining. Data are presented as mean ± SEM. Each dot represents an individual animal. N = 3-5 per genotype. *p<0.05 **p<0.001 by two-way ANOVA.

Journal: bioRxiv

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway

doi: 10.1101/2023.04.03.535167

Figure Lengend Snippet: (A) Experimental scheme of the PTS induction in WT and Slc4a4-icKO mice, followed by intraperitoneal injection of CCL2 functional blocking antibody (CCL2-FB⍺) at 1 dpi. Brains were then harvested and analyzed at 4 dpi. (B) Representative images of protein leakage (Evans blue, fibrinogen), endothelial junctional marker expression (Claudin-5+; CD31+), endothelial pCav-1 expression and reactive astrocyte marker GFAP at the peri-lesion area in WT and Slc4a4-icKO mice with or without CCL2-FB⍺ treatment. (C) Quantification of fibrinogen intensity from immunostaining. Data are presented as mean ± SEM. Each dot represents an individual animal (N = 4-9 per group). ***p<0.001, ****p<0.0001 by two-way ANOVA. (D) Quantification of CD31 intensity from immunostaining. Data are presented as mean ± SEM. n = 2-3 blood vessels per animal and N = 4-5 animals per group. **p<0.01 ***p<0.001, ****p<0.0001 by two-way ANOVA. (E) Quantification of the intensity of Claudin-5 colocalized with CD31. Data are presented as mean ± SEM. n = 2-3 blood vessels per animal and N = 4-5 animals per group. *p<0.05, ***p<0.001 by two-way ANOVA. (F) Quantification of the intensity of pCav-1 colocalized with CD31. Data are presented as mean ± SEM. n = 4-5 blood vessels per animal and N = 4-5 animals per group. *p<0.05, ***p<0.001 by two-way ANOVA. (G) Quantification of GFAP intensity from immunostaining. Data are presented as mean ± SEM. Each dot represents an individual animal. N = 3-5 per genotype. *p<0.05 **p<0.001 by two-way ANOVA.

Techniques: Injection, Functional Assay, Blocking Assay, Marker, Expressing, Immunostaining

( A-B ) WT and Slc4a4-icKO mice were intraperitoneally injected with iNOS inhibitor (L-NMMA, 10mg/kg) from 1-3 dpi. Brains were harvested at 4 dpi for analysis of cortical arginine metabolites. Data are presented as mean ± SEM. N = 4-7 animals per group. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA. (C) Total NO levels in stroked cortices were measured by the total concentration of nitrite and nitrate using a colorimetric assay. *p<0.05 by one-way ANOVA. (D-G) Representative images and quantification of CCL2 colocalized with Aldh1l1-GFP, Claudin-5 colocalized with CD31, and pCav-1 colocalized with CD31 at the peri-lesion area. Data are presented as mean ± SEM. Each data point represents individual images from multiple animals. N = 3-4 animals per group. *p<0.05, **p<0.01, ****p<0.0001 by one-way ANOVA.

Journal: bioRxiv

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway

doi: 10.1101/2023.04.03.535167

Figure Lengend Snippet: ( A-B ) WT and Slc4a4-icKO mice were intraperitoneally injected with iNOS inhibitor (L-NMMA, 10mg/kg) from 1-3 dpi. Brains were harvested at 4 dpi for analysis of cortical arginine metabolites. Data are presented as mean ± SEM. N = 4-7 animals per group. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA. (C) Total NO levels in stroked cortices were measured by the total concentration of nitrite and nitrate using a colorimetric assay. *p<0.05 by one-way ANOVA. (D-G) Representative images and quantification of CCL2 colocalized with Aldh1l1-GFP, Claudin-5 colocalized with CD31, and pCav-1 colocalized with CD31 at the peri-lesion area. Data are presented as mean ± SEM. Each data point represents individual images from multiple animals. N = 3-4 animals per group. *p<0.05, **p<0.01, ****p<0.0001 by one-way ANOVA.

Techniques: Injection, Concentration Assay, Colorimetric Assay

Journal: bioRxiv

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway

doi: 10.1101/2023.04.03.535167

Figure Lengend Snippet: (A) Conditioned media (CM) was collected from primary WT and Slc4a4 KO astrocytes and subjected to LC-MS/MS-based unbiased proteomics and cytokine/chemokine array. (B) Angiogenic factors detected from LC-MS/MS-based unbiased proteomics. (C) Cytokine/chemokines that are changed in the CM from WT and Slc4a4 KO astrocytes. (D) CM from primary WT and Slc4a4 KO astrocytes cultured under either normal or oxygen-glucose deprivation (OGD) condition was collected for CCL2 measurement by ELISA. Each dot represents conditioned media collected from individual animals’ astrocytes. N = 3–5 per genotype. *p<0.05, ****p<0.01 by Student’s t-test. (E) Proposed model for the Slc4a4-CCL2-CCR2 axis regulating astrocyte-endothelia interaction. (F) Quantification of cortical CCL2 levels in the uninjured and stroked (1 dpi) brains from WT and Slc4a4-icKO mice. Data are presented as mean ± SEM. N = 3–5 mice per genotype. *p<0.05, **p<0.01 by Student’s t-test. (G) Quantification of astrocytic CCL2 expression from double immunostaining. Data are presented as mean ± SEM. n = 7–17 cells collected from N = 3–5 mice per genotype. *p<0.05 by Student’s t-test. (H) Quantification of endothelial CCR2 expression from double immunostaining. Data are presented as mean ± SEM. Each dot represents an individual animal. N = 4–5 per genotype. *p<0.05, *p<0.05 by Student’s t-test. (I) At 4 dpi, astrocytic CCL2 expression was visualized by double immunostaining of CCL2/GFAP. Endothelial and astrocytic CCR2 expression was visualized by double immunostaining of CCR2/CD31 and CCR2/Aldh1l1-GFP respectively.

Article Snippet: The following commercial primary antibodies were used: rabbit anti-GFAP (1:1,000; Agilent Dako), mouse anti-NeuN (1:500; Millipore), rabbit anti-GFP (1:1,000; Chromotek; PABG1-100), rabbit anti-NF1A (gift from Dr. Benjamin Deneen), rabbit anti-Sox9 (1:1,000; Millipore; AB5535), rat anti-BrdU (1:1,000; Abcam; ab6326), rabbit anti-S100b (1:1,000; Agilent Dako; Z0311), Rat anti-CD31(1:200; BD Bioscience; 55047), rabbit anti-Glut1 (1:500; Millipore; 07–1401), rabbit anti-AQP4(1:500; Sigma; A5971), rabbit anti-ZO-1 (1:250; Invitrogen;40–2200), Alexa 488 conjugated Claudin-5 (1:500; Invitrogen, 35-358-8), rabbit anti-CCL2 (1:500, PerproTech, 500-P113), goat anti-CCL2 (1:500; R&D, AF-479), ), rabbit anit-CCR2 (1:500; Abcam; ab273050), mouse anti-Cav-1 (1:500; BD Bioscience; 610406), mouse anti-pCav-1 (1:500; BD Bioscience; 611339).

Techniques: Liquid Chromatography with Mass Spectroscopy, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Double Immunostaining

Journal: bioRxiv

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway

doi: 10.1101/2023.04.03.535167

Figure Lengend Snippet: (A) Mouse endothelial cells (bEnd3) were incubated with conditioned media (CM) collected from primary WT and Slc4a4 KO astrocytes with CCL2 functional blocking antibody (CCL2 αFB, 15 ng/ml) or CCR2 antagonist for 24 hours. (B-D) Representative immunofluorescence images and quantification of tight junction proteins (ZO-1 and Claudin-5) of bEnd3 cells incubated with WT- or Slc4a4 KO- CM in the presence of control IgG or CCL2-FBα. Data are presented as mean ± SEM. Each dot represents a well collected from 3 independent experiments. *p<0.05, ***p<0.001 by two-way ANOVA. (E) Experimental setup of the transendothelial electrical resistance (TEER) assay. A monolayer of bEnd3 cells was co-cultured with WT or Slc4a4-deficient astrocytes, where astrocytes were plated at the bottom of the well and endothelial cells were plated in the insert, allowing endothelial cells to be exposed to astrocytic secreting factors without physical contact with astrocytes. CCL2 blocking antibody was added into the insert with a final concentration of 15 ng/ml. (F) The electro-resistance of the endothelial cell monolayer was measured as an indicator for the permeability of endothelial cells in the TEER assay. Data are presented as mean ± SEM. Each dot represents each independent culture. N = 4 independent assays. *p<0.05, **p<0.01 by two-way ANOVA. (G-H) Caveolin- and clathrin-mediated endothelial intracellular uptake was examined by Texas Red conjugated albumin and A488-transferrin, respectively, in bEnd3 cells incubated with WT- or Slc4a4 KO-CM with CCR2 FBα or control IgG. Data are presented as mean ± SEM. Each dot represents each independent culture. N= 3 independent assays. **p<0.01, ***p<0.001 by two-way ANOVA. (I-J) Western blot analysis of pCav-1, Cav-1 and CCR2 expression in bEnd3 cells incubated with WT- or Slc4a4 KO-CM with CCR2 FBα or control IgG. Data are presented as mean ± SEM. Each dot represents each independent culture. N = 3 independent assays. **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA. (K-L) Paracellular and transcellular endothelial transport in bEnd3 cells incubated with WT- or Slc4a4 KO-CM with CCR2 antagonist RS504393 (10 μM) or control DMSO were examined by immunostaining of ZO-1 and pCav-1. Data are presented as mean ± SEM. Each dot represents each independent culture. N = 3 independent assays. *p<0.05, **p<0.01, ***p<0.001 by two-way ANOVA. (M) A proposed mechanism by which astrocytic Slc4a4 regulates endothelial paracellular and transcellular transport pathways via the CCL2-CCR2 axis.

Article Snippet: The following commercial primary antibodies were used: rabbit anti-GFAP (1:1,000; Agilent Dako), mouse anti-NeuN (1:500; Millipore), rabbit anti-GFP (1:1,000; Chromotek; PABG1-100), rabbit anti-NF1A (gift from Dr. Benjamin Deneen), rabbit anti-Sox9 (1:1,000; Millipore; AB5535), rat anti-BrdU (1:1,000; Abcam; ab6326), rabbit anti-S100b (1:1,000; Agilent Dako; Z0311), Rat anti-CD31(1:200; BD Bioscience; 55047), rabbit anti-Glut1 (1:500; Millipore; 07–1401), rabbit anti-AQP4(1:500; Sigma; A5971), rabbit anti-ZO-1 (1:250; Invitrogen;40–2200), Alexa 488 conjugated Claudin-5 (1:500; Invitrogen, 35-358-8), rabbit anti-CCL2 (1:500, PerproTech, 500-P113), goat anti-CCL2 (1:500; R&D, AF-479), ), rabbit anit-CCR2 (1:500; Abcam; ab273050), mouse anti-Cav-1 (1:500; BD Bioscience; 610406), mouse anti-pCav-1 (1:500; BD Bioscience; 611339).

Techniques: Incubation, Functional Assay, Blocking Assay, Immunofluorescence, Control, Cell Culture, Concentration Assay, Permeability, Western Blot, Expressing, Immunostaining

Journal: bioRxiv

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway

doi: 10.1101/2023.04.03.535167

Figure Lengend Snippet: (A) Experimental scheme of the PTS induction in WT and Slc4a4-icKO mice, followed by intraperitoneal injection of CCL2 functional blocking antibody (CCL2-FBα) at 1 dpi. Brains were then harvested and analyzed at 4 dpi. (B) Representative images of protein leakage (Evans blue, fibrinogen), endothelial junctional marker expression (Claudin-5+; CD31+), endothelial pCav-1 expression and reactive astrocyte marker GFAP at the peri-lesion area in WT and Slc4a4-icKO mice with or without CCL2-FBα treatment. (C) Quantification of fibrinogen intensity from immunostaining. Data are presented as mean ± SEM. Each dot represents an individual animal (N = 4–9 per group). ***p<0.001, ****p<0.0001 by two-way ANOVA. (D) Quantification of CD31 intensity from immunostaining. Data are presented as mean ± SEM. n = 2–3 blood vessels per animal and N = 4–5 animals per group. **p<0.01 ***p<0.001, ****p<0.0001 by two-way ANOVA. (E) Quantification of the intensity of Claudin-5 colocalized with CD31. Data are presented as mean ± SEM. n = 2–3 blood vessels per animal and N = 4–5 animals per group. *p<0.05, ***p<0.001 by two-way ANOVA. (F) Quantification of the intensity of pCav-1 colocalized with CD31. Data are presented as mean ± SEM. n = 4–5 blood vessels per animal and N = 4–5 animals per group. *p<0.05, ***p<0.001 by two-way ANOVA. (G) Quantification of GFAP intensity from immunostaining. Data are presented as mean ± SEM. Each dot represents an individual animal. N = 3–5 per genotype. *p<0.05 **p<0.001 by two-way ANOVA.

Article Snippet: The following commercial primary antibodies were used: rabbit anti-GFAP (1:1,000; Agilent Dako), mouse anti-NeuN (1:500; Millipore), rabbit anti-GFP (1:1,000; Chromotek; PABG1-100), rabbit anti-NF1A (gift from Dr. Benjamin Deneen), rabbit anti-Sox9 (1:1,000; Millipore; AB5535), rat anti-BrdU (1:1,000; Abcam; ab6326), rabbit anti-S100b (1:1,000; Agilent Dako; Z0311), Rat anti-CD31(1:200; BD Bioscience; 55047), rabbit anti-Glut1 (1:500; Millipore; 07–1401), rabbit anti-AQP4(1:500; Sigma; A5971), rabbit anti-ZO-1 (1:250; Invitrogen;40–2200), Alexa 488 conjugated Claudin-5 (1:500; Invitrogen, 35-358-8), rabbit anti-CCL2 (1:500, PerproTech, 500-P113), goat anti-CCL2 (1:500; R&D, AF-479), ), rabbit anit-CCR2 (1:500; Abcam; ab273050), mouse anti-Cav-1 (1:500; BD Bioscience; 610406), mouse anti-pCav-1 (1:500; BD Bioscience; 611339).

Techniques: Injection, Functional Assay, Blocking Assay, Marker, Expressing, Immunostaining

Journal: bioRxiv

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway

doi: 10.1101/2023.04.03.535167

Figure Lengend Snippet: ( A-B ) WT and Slc4a4-icKO mice were intraperitoneally injected with iNOS inhibitor (L-NMMA, 10mg/kg) from 1–3 dpi. Brains were harvested at 4 dpi for analysis of cortical arginine metabolites. Data are presented as mean ± SEM. N = 4–7 animals per group. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA. (C) Total NO levels in stroked cortices were measured by the total concentration of nitrite and nitrate using a colorimetric assay. *p<0.05 by one-way ANOVA. (D-G) Representative images and quantification of CCL2 colocalized with Aldh1l1-GFP, Claudin-5 colocalized with CD31, and pCav-1 colocalized with CD31 at the peri-lesion area. Data are presented as mean ± SEM. Each data point represents individual images from multiple animals. N = 3–4 animals per group. *p<0.05, **p<0.01, ****p<0.0001 by one-way ANOVA.

Article Snippet: The following commercial primary antibodies were used: rabbit anti-GFAP (1:1,000; Agilent Dako), mouse anti-NeuN (1:500; Millipore), rabbit anti-GFP (1:1,000; Chromotek; PABG1-100), rabbit anti-NF1A (gift from Dr. Benjamin Deneen), rabbit anti-Sox9 (1:1,000; Millipore; AB5535), rat anti-BrdU (1:1,000; Abcam; ab6326), rabbit anti-S100b (1:1,000; Agilent Dako; Z0311), Rat anti-CD31(1:200; BD Bioscience; 55047), rabbit anti-Glut1 (1:500; Millipore; 07–1401), rabbit anti-AQP4(1:500; Sigma; A5971), rabbit anti-ZO-1 (1:250; Invitrogen;40–2200), Alexa 488 conjugated Claudin-5 (1:500; Invitrogen, 35-358-8), rabbit anti-CCL2 (1:500, PerproTech, 500-P113), goat anti-CCL2 (1:500; R&D, AF-479), ), rabbit anit-CCR2 (1:500; Abcam; ab273050), mouse anti-Cav-1 (1:500; BD Bioscience; 610406), mouse anti-pCav-1 (1:500; BD Bioscience; 611339).

Techniques: Injection, Concentration Assay, Colorimetric Assay

Figure 6. Effects of OSM on the protein expression of exercise-induced chemokines in the skeletal muscle. A, Western blot analysis of CCL2, CCL7, and CXCL1 in the skeletal muscle of sedentary mice (Sed [1 h] + 2 h) and mice at 2 h after the exercise (Ex [1 h] + 2 h). The apparent molecular weights are indicated on the right. B, quantitative analysis of the protein expression of CCL2, CCL7, and CXCL1. The band intensities of CCL2, CCL7, and CXCL1 were normalized to β-tubulin and are represented as the fold induction relative to the intensities of sedentary mice in the bar graph. C, Western blot analysis of CCL2, CCL7, and CXCL1 in the skeletal muscle of WT and OSM−/−mice at 2 h after the exercise. The apparent molecular weights are indicated on the right. D, quantitative analysis of the protein expression of CCL2, CCL7, and CXCL1. The band intensities of CCL2, CCL7, and CXCL1 were normalized to β-tubulin and are represented as the fold induction relative to the intensities of WT mice in the bar graph. E, Western blot analysis of CCL2, CCL7, and CXCL1 in the skeletal muscle of mice at 2 h after the injection of vehicle (Veh) or OSM. F, quantitative analysis of the protein expression of CCL2, CCL7, and CXCL1. The band intensities of CCL2, CCL7, and CXCL1 were normalized to β-tubulin and are represented as the fold induction relative to the intensities of Veh group in the bar graph. Data are expressed as mean ± SD; n = 5 per group. *p < 0.05, **p < 0.01, and ***p < 0.001. Student’s t test. CCL, CC chemokine ligand; CXCL1, CXC chemokine ligand 1; OSM, oncostatin M.

Journal: The Journal of biological chemistry

Article Title: Essential roles of the cytokine oncostatin M in crosstalk between muscle fibers and immune cells in skeletal muscle after aerobic exercise.

doi: 10.1016/j.jbc.2022.102686

Figure Lengend Snippet: Figure 6. Effects of OSM on the protein expression of exercise-induced chemokines in the skeletal muscle. A, Western blot analysis of CCL2, CCL7, and CXCL1 in the skeletal muscle of sedentary mice (Sed [1 h] + 2 h) and mice at 2 h after the exercise (Ex [1 h] + 2 h). The apparent molecular weights are indicated on the right. B, quantitative analysis of the protein expression of CCL2, CCL7, and CXCL1. The band intensities of CCL2, CCL7, and CXCL1 were normalized to β-tubulin and are represented as the fold induction relative to the intensities of sedentary mice in the bar graph. C, Western blot analysis of CCL2, CCL7, and CXCL1 in the skeletal muscle of WT and OSM−/−mice at 2 h after the exercise. The apparent molecular weights are indicated on the right. D, quantitative analysis of the protein expression of CCL2, CCL7, and CXCL1. The band intensities of CCL2, CCL7, and CXCL1 were normalized to β-tubulin and are represented as the fold induction relative to the intensities of WT mice in the bar graph. E, Western blot analysis of CCL2, CCL7, and CXCL1 in the skeletal muscle of mice at 2 h after the injection of vehicle (Veh) or OSM. F, quantitative analysis of the protein expression of CCL2, CCL7, and CXCL1. The band intensities of CCL2, CCL7, and CXCL1 were normalized to β-tubulin and are represented as the fold induction relative to the intensities of Veh group in the bar graph. Data are expressed as mean ± SD; n = 5 per group. *p < 0.05, **p < 0.01, and ***p < 0.001. Student’s t test. CCL, CC chemokine ligand; CXCL1, CXC chemokine ligand 1; OSM, oncostatin M.

Article Snippet: Primary antibodies were used at the following dilutions: rabbit anti-iNOS antibody (diluted at 1:2000; catalog no.: ab15323, Abcam); rabbit anti-TNF-α antibody (diluted at 1:2000; catalog no.: ab6671, Abcam); sheep anti-arginase-1 antibody (diluted at 1:2000; catalog no.: AF5868, R&D Systems); goat anti-IL-10 antibody (diluted at 1:1000; catalog no.: AF519, R&D Systems); goat anti-OSM antibody (diluted at 1:1000, R&D Systems); goat anti-CCL2 antibody (diluted at 1:1000, catalog no.: AF-479-NA, R&D Systems); goat anti-CCL7 antibody (diluted at 1:1000, catalog no.: AF-456-NA, R&D Systems); goat anti-CXCL1 antibody (diluted at 1:1000, catalog no.: AF-453-NA, R&D Systems); goat anti-OSMRβ antibody (diluted at 1:1000, R&D Systems); rabbit anti–phospho-STAT3 antibody (diluted at 1:1000); and rabbit anti–phospho-Akt antibody (diluted 1:2000, catalog no.: 4060, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Injection

Secretion of S100A8/A9 by pro-inflammatory monocytes is under control of CCL2. ( a ) S100A8/A9 concentration in supernatant of FACS-sorted CCR2 + CX3CR1 low pro-inflammatory monocytes (n=5) and Gr-1 + CD115 neg (n=4) from spleens of 67NR and 4T1.2 tumor-bearing mice stimulated 48h with recombinant mouse CCL2. S100A8/A9 concentration was determined by ELISA in two independent experiments. ( b ) Axial and coronal images from SPECT analysis and relative in vivo tracer uptake in 4T1.2 tumor-bearing mice treated with an isotype control antibody (IgG) or a blocking anti-CCL2 antibody. The frequency of CCR2 + CX3CR1 low and average relative number of CCR2 + CX3CR1 low /10 6 live cells in spleens ( c ) and lungs ( d ) (n=4, for each organ and treatment analysed in two independent experiments 14d after tumor induction) is reduced under CCL2-blockade. Mean ± SE: * p <0.05.

Journal: Theranostics

Article Title: Visualization of Tumor-Immune Interaction - Target-Specific Imaging of S100A8/A9 Reveals Pre-Metastatic Niche Establishment

doi: 10.7150/thno.17138

Figure Lengend Snippet: Secretion of S100A8/A9 by pro-inflammatory monocytes is under control of CCL2. ( a ) S100A8/A9 concentration in supernatant of FACS-sorted CCR2 + CX3CR1 low pro-inflammatory monocytes (n=5) and Gr-1 + CD115 neg (n=4) from spleens of 67NR and 4T1.2 tumor-bearing mice stimulated 48h with recombinant mouse CCL2. S100A8/A9 concentration was determined by ELISA in two independent experiments. ( b ) Axial and coronal images from SPECT analysis and relative in vivo tracer uptake in 4T1.2 tumor-bearing mice treated with an isotype control antibody (IgG) or a blocking anti-CCL2 antibody. The frequency of CCR2 + CX3CR1 low and average relative number of CCR2 + CX3CR1 low /10 6 live cells in spleens ( c ) and lungs ( d ) (n=4, for each organ and treatment analysed in two independent experiments 14d after tumor induction) is reduced under CCL2-blockade. Mean ± SE: * p <0.05.

Article Snippet: For CCL2 blocking, 4T1.2 tumor-bearing mice received an intraperitoneal injection of 100µg goat polyclonal anti-CCL2 antibody (R&D, Abingdon, UK) every other day beginning on day 4 after tumor inoculation, following established protocols .

Techniques: Concentration Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Single Photon Emission Computed Tomography, In Vivo, Blocking Assay

S100A8/A9-induced immune remodelling in lungs of mice 4T1.2-tumor bearing mice predicts metastasis. ( a ) Exemplary axial and coronal in vivo images from SPECT examination and relative tracer accumulation graphs of healthy control animals and 67NR or 4T1.2 tumor-bearing mice (d10 after tumor induction) after injection of the S100A8/A9-specific tracer or unspecific IgG to control for perfusion effects. While the unspecific IgG does not show any differences between the three groups, S100A9-SPECT reveals ongoing monocytes activation and immune remodelling in the 4T1.2 tumor-bearing mice, reflected by a strong tracer-accumulation. ( b ) Frequency of Gr-1 + CD115 + CCR2 + CX3CR1 low monocytes in lungs of control non tumor-bearing mice, 67NR and 4T1.2 tumor-bearing mice. The bar graph shows average frequency of Gr-1 + CD115 + and the relative number of CCR2 + CX3CR1 low /10 6 live cells from 5 independent experiments. ( c ) Expression of CD115 (blue), CCR2 (magenta) and S100A8/A9 (yellow) in frozen lung sections from 4T1.2-tumor bearing mice (n=3). Extracellular S100 signal is indicated by white arrows. ( d ) Representative plots and bar graph showing the frequency of mCherry + 4T1.2 cells in the lungs of 4T1.2 tumor-bearing mice at 10 and 20 days after tumor induction (n=4, one of two experiments shown). ( e ) Correlation between S100A8/A9 activity in the lungs of 4T1.2 tumor-bearing mice at day 10 and the frequency of mCherry + 4T1.2 tumor cells at day 21 after tumor induction (n=9). Red dots indicate animals that received anti CCL2 treatment. Mean ± SE: *** p <0.005, * p <0.05.

Journal: Theranostics

Article Title: Visualization of Tumor-Immune Interaction - Target-Specific Imaging of S100A8/A9 Reveals Pre-Metastatic Niche Establishment

doi: 10.7150/thno.17138

Figure Lengend Snippet: S100A8/A9-induced immune remodelling in lungs of mice 4T1.2-tumor bearing mice predicts metastasis. ( a ) Exemplary axial and coronal in vivo images from SPECT examination and relative tracer accumulation graphs of healthy control animals and 67NR or 4T1.2 tumor-bearing mice (d10 after tumor induction) after injection of the S100A8/A9-specific tracer or unspecific IgG to control for perfusion effects. While the unspecific IgG does not show any differences between the three groups, S100A9-SPECT reveals ongoing monocytes activation and immune remodelling in the 4T1.2 tumor-bearing mice, reflected by a strong tracer-accumulation. ( b ) Frequency of Gr-1 + CD115 + CCR2 + CX3CR1 low monocytes in lungs of control non tumor-bearing mice, 67NR and 4T1.2 tumor-bearing mice. The bar graph shows average frequency of Gr-1 + CD115 + and the relative number of CCR2 + CX3CR1 low /10 6 live cells from 5 independent experiments. ( c ) Expression of CD115 (blue), CCR2 (magenta) and S100A8/A9 (yellow) in frozen lung sections from 4T1.2-tumor bearing mice (n=3). Extracellular S100 signal is indicated by white arrows. ( d ) Representative plots and bar graph showing the frequency of mCherry + 4T1.2 cells in the lungs of 4T1.2 tumor-bearing mice at 10 and 20 days after tumor induction (n=4, one of two experiments shown). ( e ) Correlation between S100A8/A9 activity in the lungs of 4T1.2 tumor-bearing mice at day 10 and the frequency of mCherry + 4T1.2 tumor cells at day 21 after tumor induction (n=9). Red dots indicate animals that received anti CCL2 treatment. Mean ± SE: *** p <0.005, * p <0.05.

Techniques: In Vivo, Single Photon Emission Computed Tomography, Injection, Activation Assay, Expressing, Activity Assay

Serum inflammatory cytokine concentrations in DE50-MD and WT dogs. Comparison of Luminex assay results for DE50-MD (grey) and WT (white) dog serum concentrations for (A) CCL2, (B) GM-CSF, (C) KC-like protein, (D) TNFα, (E) IFNγ and (F) IP-10. Dogs were studied longitudinally between 3 and 18 months of age. Month 3: WT, n =11; DE50-MD, n =12. Month 6: WT, n =11; DE50-MD, n =12. Month 9: WT, n =11; DE50-MD, n =8. Month 12: WT, n =11; DE50-MD, n =7. Month 15: WT, n =9; DE50-MD, n =6. Month 18: WT, n =9; DE50-MD, n =5. Boxes extend from the 25th to 75th percentile, with a line within the box at the median value. Each point represents an individual dog, and whiskers show the minimum and maximum results for that age group. Results that were below the lower limit of detection of the assay are presented as the minimum detectable concentration. Concentrations varied over orders of magnitudes between and/or within groups for some of the cytokines measured; therefore, a logarithmic scale was used on the y -axis to better display the data for graphs A,B,D and F. Asterisks indicate significance level based on linear mixed-model analysis, adjusted for repeated measures. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001.

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Serum inflammatory cytokine concentrations in DE50-MD and WT dogs. Comparison of Luminex assay results for DE50-MD (grey) and WT (white) dog serum concentrations for (A) CCL2, (B) GM-CSF, (C) KC-like protein, (D) TNFα, (E) IFNγ and (F) IP-10. Dogs were studied longitudinally between 3 and 18 months of age. Month 3: WT, n =11; DE50-MD, n =12. Month 6: WT, n =11; DE50-MD, n =12. Month 9: WT, n =11; DE50-MD, n =8. Month 12: WT, n =11; DE50-MD, n =7. Month 15: WT, n =9; DE50-MD, n =6. Month 18: WT, n =9; DE50-MD, n =5. Boxes extend from the 25th to 75th percentile, with a line within the box at the median value. Each point represents an individual dog, and whiskers show the minimum and maximum results for that age group. Results that were below the lower limit of detection of the assay are presented as the minimum detectable concentration. Concentrations varied over orders of magnitudes between and/or within groups for some of the cytokines measured; therefore, a logarithmic scale was used on the y -axis to better display the data for graphs A,B,D and F. Asterisks indicate significance level based on linear mixed-model analysis, adjusted for repeated measures. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001.

Article Snippet: Sections were then incubated with the following combinations of primary antibodies for 1 h at room temperature: goat anti-canine CCL2 IgG (R&D systems, AF1774, 1:20) with rat anti-human perlecan (Millipore, clone A7L6, 1:1000) , mouse anti-canine CD18 (Bio-Rad, monoclonal IgG1, clone CA1.4E9, MA1-82363, 1:100) ( C) or mouse anti-human macrophages (Biorad, monoclonal IgG1, clone MAC387, MCA874GT, 1:100) ( D).

Techniques: Comparison, Luminex, Concentration Assay

RT-qPCR of CCL2 mRNA in vastus lateralis muscle of DE50-MD and WT dogs. Results were normalised to the expression of three reference genes: RPL13A , HPRT1 and SDHA . Boxes extend from the 25th to 75th percentile, with a line within the box at the median value. Each point represents an individual DE50-MD or WT dog studied longitudinally between 3 and 18 months of age, and whiskers show the minimum and maximum results for that age group. Month 3: WT, n =4; DE50-MD, n =9. Month 6: WT, n =10; DE50-MD, n =10. Month 9: WT, n =10; DE50-MD, n =7. Month 12: WT, n =9; DE50-MD, n =6. Month 15: WT, n =8; DE50-MD, n =5. Month 18: WT, n =9; DE50-MD, n =6. Asterisks denote the level of significance of a difference between genotypes based on linear mixed-model analysis, adjusted for repeated measures. * P <0.05; ** P <0.01.

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: RT-qPCR of CCL2 mRNA in vastus lateralis muscle of DE50-MD and WT dogs. Results were normalised to the expression of three reference genes: RPL13A , HPRT1 and SDHA . Boxes extend from the 25th to 75th percentile, with a line within the box at the median value. Each point represents an individual DE50-MD or WT dog studied longitudinally between 3 and 18 months of age, and whiskers show the minimum and maximum results for that age group. Month 3: WT, n =4; DE50-MD, n =9. Month 6: WT, n =10; DE50-MD, n =10. Month 9: WT, n =10; DE50-MD, n =7. Month 12: WT, n =9; DE50-MD, n =6. Month 15: WT, n =8; DE50-MD, n =5. Month 18: WT, n =9; DE50-MD, n =6. Asterisks denote the level of significance of a difference between genotypes based on linear mixed-model analysis, adjusted for repeated measures. * P <0.05; ** P <0.01.

Techniques: Quantitative RT-PCR, Expressing

Relationship between serum CCL2 protein and vastus lateralis muscle CCL2 mRNA. Graph relationships between the relative levels of CCL2 mRNA in the vastus lateralis muscle and serum CCL2 protein concentration (A), and between the relative CCL2 and CCR2 mRNA levels in the vastus lateralis muscle (B) of DE50-MD (black triangles) and WT (open circles). No relationship was found between serum CCL2 protein concentration and vastus lateralis muscle CCL2 mRNA levels within genotypes (DE50-MD: P =0.59, n =36 samples from a total of 13 dogs; WT: P =0.95, n =45 samples from a total of 11 dogs). A positive association between CCL2 and CCR2 mRNA levels in the vastus lateralis muscle was found for both WT ( P <0.0001, n =49 samples from a total of 11 dogs) and DE50-MD ( P =0.001, n =42 samples from a total of 13 dogs) subjects, based on linear mixed-model analysis. Repeated measures were accounted for in the statistical analyses.

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Relationship between serum CCL2 protein and vastus lateralis muscle CCL2 mRNA. Graph relationships between the relative levels of CCL2 mRNA in the vastus lateralis muscle and serum CCL2 protein concentration (A), and between the relative CCL2 and CCR2 mRNA levels in the vastus lateralis muscle (B) of DE50-MD (black triangles) and WT (open circles). No relationship was found between serum CCL2 protein concentration and vastus lateralis muscle CCL2 mRNA levels within genotypes (DE50-MD: P =0.59, n =36 samples from a total of 13 dogs; WT: P =0.95, n =45 samples from a total of 11 dogs). A positive association between CCL2 and CCR2 mRNA levels in the vastus lateralis muscle was found for both WT ( P <0.0001, n =49 samples from a total of 11 dogs) and DE50-MD ( P =0.001, n =42 samples from a total of 13 dogs) subjects, based on linear mixed-model analysis. Repeated measures were accounted for in the statistical analyses.

Techniques: Protein Concentration

$Relationship between serum CCL2 protein concentration and other biomarkers of disease in DE50-MD dogs. (A) Graph relationship between serum CCL2 concentration and plasma CK activity. A positive relationship was found between the two variables (R 2 =0.37, P <0.0001). n =41 samples, from a total of 14 DE50-MD dogs studied longitudinally between 3 and 18 months of age (month 3, n =11; month 6, n =9; month 9, n =7; month 12, n =5; month 15, n =5; month 18, n =4). (B) Graph relationship between serum CCL2 concentration and the geometric mean (geomean) of acid phosphatase-stained fraction of vastus lateralis muscle biopsy samples. A positive relationship was found between the two variables in DE50-MD dogs (R 2 =0.41, P =0.0008). n =42 samples, from a total of 14 DE50-MD dogs studied longitudinally between 3 and 18 months of age (month 3, n =7; month 6, n =10; month 9, n =8; month 12, n =7; month 15, n =5; month 18, n =5). Linear regressions were calculated based on linear mixed-model analysis, accounting for repeated measures.$

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Relationship between serum CCL2 protein concentration and other biomarkers of disease in DE50-MD dogs. (A) Graph relationship between serum CCL2 concentration and plasma CK activity. A positive relationship was found between the two variables (R 2 =0.37, P <0.0001). n =41 samples, from a total of 14 DE50-MD dogs studied longitudinally between 3 and 18 months of age (month 3, n =11; month 6, n =9; month 9, n =7; month 12, n =5; month 15, n =5; month 18, n =4). (B) Graph relationship between serum CCL2 concentration and the geometric mean (geomean) of acid phosphatase-stained fraction of vastus lateralis muscle biopsy samples. A positive relationship was found between the two variables in DE50-MD dogs (R 2 =0.41, P =0.0008). n =42 samples, from a total of 14 DE50-MD dogs studied longitudinally between 3 and 18 months of age (month 3, n =7; month 6, n =10; month 9, n =8; month 12, n =7; month 15, n =5; month 18, n =5). Linear regressions were calculated based on linear mixed-model analysis, accounting for repeated measures.

Techniques: Protein Concentration, Concentration Assay, Clinical Proteomics, Activity Assay, Staining

Histology of the vastus lateralis muscle from WT and DE50-MD littermates from 3 to 18 months of age. (A,B) Immunohistochemistry (IHC) of vastus lateralis muscle from (A) a WT dog (ID WT-T3) and (B) a DE50-MD dog (ID DE50-T6). Each section was labelled with antibodies against perlecan (green) and CCL2 (magenta), and nuclei were labelled with Hoechst 33342 (blue). (C,D) For the DE50-MD dog DE50-T6, serial sections for each timepoint were stained for (C) acid phosphatase (AP) and with (D) H&E. Scale bar: 100 µm.

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Histology of the vastus lateralis muscle from WT and DE50-MD littermates from 3 to 18 months of age. (A,B) Immunohistochemistry (IHC) of vastus lateralis muscle from (A) a WT dog (ID WT-T3) and (B) a DE50-MD dog (ID DE50-T6). Each section was labelled with antibodies against perlecan (green) and CCL2 (magenta), and nuclei were labelled with Hoechst 33342 (blue). (C,D) For the DE50-MD dog DE50-T6, serial sections for each timepoint were stained for (C) acid phosphatase (AP) and with (D) H&E. Scale bar: 100 µm.

Techniques: Immunohistochemistry, Staining

Histology of the vastus lateralis muscle from a 6-month-old DE50-MD dog. (A-E) Immunohistochemistry of a section of vastus lateralis muscle showing (A) CCL2 (magenta), (B) CD18 (green) and (C) a merged image of CCL2, CD18 and Hoechst 33342 (blue), with magnified views of the boxed region in C shown for (D) CCL2 and (E) CD18 staining. (F-J) Immunohistochemistry of a serial section showing (F) CCL2 (magenta), (G) MAC387 + regions (green) and (H) a merged image of CCL2, MAC387 and Hoechst 33342 (blue), with magnified views of the boxed region in H shown for (I) CCL2 and (J) CD18 staining. (K) A serial section with H&E staining showing cell infiltrate within and between muscle fibres, fibres with internalised nuclei and hypercontracted fibres (darker magenta). (L) Serial section with acid phosphatase (AP) staining showing positive red staining at areas of cell infiltrate and within some muscle fibres. (M-O) Serial section of immunohistochemistry with no primary antibody as a control with magnified views of the region of inflammation (dashed box) shown for the (N) magenta (CCL2) and (O) green (CD18/MAC387) channels. Scale bar: 100 µm.

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Histology of the vastus lateralis muscle from a 6-month-old DE50-MD dog. (A-E) Immunohistochemistry of a section of vastus lateralis muscle showing (A) CCL2 (magenta), (B) CD18 (green) and (C) a merged image of CCL2, CD18 and Hoechst 33342 (blue), with magnified views of the boxed region in C shown for (D) CCL2 and (E) CD18 staining. (F-J) Immunohistochemistry of a serial section showing (F) CCL2 (magenta), (G) MAC387 + regions (green) and (H) a merged image of CCL2, MAC387 and Hoechst 33342 (blue), with magnified views of the boxed region in H shown for (I) CCL2 and (J) CD18 staining. (K) A serial section with H&E staining showing cell infiltrate within and between muscle fibres, fibres with internalised nuclei and hypercontracted fibres (darker magenta). (L) Serial section with acid phosphatase (AP) staining showing positive red staining at areas of cell infiltrate and within some muscle fibres. (M-O) Serial section of immunohistochemistry with no primary antibody as a control with magnified views of the region of inflammation (dashed box) shown for the (N) magenta (CCL2) and (O) green (CD18/MAC387) channels. Scale bar: 100 µm.

Techniques: Immunohistochemistry, Staining, Control

Samples tested for each study dog. (A) Serum samples tested by Luminex assay and (B) vastus lateralis muscle samples assayed by RT-qPCR for quantification of CCL2 and CCR2 mRNA. Age is displayed in months. Dog ID shows the study name of each individual animal included in this study: dog IDs with the prefix ‘DE50-‘ are DE50-MD dogs, dogs with the prefix ‘WT’ are wild-type dogs. White boxes show samples that were tested. Grey boxes indicate untested samples: dark grey boxes indicate samples that were unavailable due to animal euthanasia prior to this timepoint, whereas light grey boxes indicate samples unavailable for other reasons.

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Samples tested for each study dog. (A) Serum samples tested by Luminex assay and (B) vastus lateralis muscle samples assayed by RT-qPCR for quantification of CCL2 and CCR2 mRNA. Age is displayed in months. Dog ID shows the study name of each individual animal included in this study: dog IDs with the prefix ‘DE50-‘ are DE50-MD dogs, dogs with the prefix ‘WT’ are wild-type dogs. White boxes show samples that were tested. Grey boxes indicate untested samples: dark grey boxes indicate samples that were unavailable due to animal euthanasia prior to this timepoint, whereas light grey boxes indicate samples unavailable for other reasons.

Techniques: Luminex, Quantitative RT-PCR

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