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cxcl6 (Boster Bio)

Name: cxcl6
Brand: Boster Bio
Rating: 90 (6 reviews)

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Boster Bio cxcl6
Cxcl6, supplied by Boster Bio, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 6 article reviews

cxcl6 - by Bioz Stars, 2026-02

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A, B) The electrical resistance and permeability in HLMVECs after treatment with VEGF-D (1 μg/mL), adrenomedullin (ADM)(100 ng/mL), angiopoietin 1 (ANGPT1)(2 μg/mL), BMP-5 (1 μg/mL), SLIT2 (1 μg/mL), CXCL6 (1 μg/mL), Pleiotrophin (PTN)(1 μg/mL), SEMA6D (1 μg/mL), and TNF-α (1 ng/mL) were measured through electrical cell impendence sensing (ECIS) assay, and transwell assay, respectively. C) HLMVECs cultured on transwell treated with VEGF-D, followed by challenging with IL1β, TNF-α, or thrombin. The permeability was determined using a 20kDa FITC-dextran probe. D) Immunofluorescent images of F actin (red), and VE-Cadherin (green) in HLMVECs challenged with TNF-α or IL1β with or without VEGF-D. White boxes highlight VE-Cadherin junctions in all conditions. Scale bar 20 μm. E) Protein expression of phosphor-MLC in HLMVECs challenged with thrombin with different concentrations of VEGF-D was assessed through a western blot. Data presented as mean ± SEM. * and ** indicate p<0.05 and p<0.01, respectively.

Journal: bioRxiv

Article Title: Vascular endothelial growth factor-D improves lung vascular integrity during acute lung injury

doi: 10.1101/2024.12.16.628787

Figure Lengend Snippet: A, B) The electrical resistance and permeability in HLMVECs after treatment with VEGF-D (1 μg/mL), adrenomedullin (ADM)(100 ng/mL), angiopoietin 1 (ANGPT1)(2 μg/mL), BMP-5 (1 μg/mL), SLIT2 (1 μg/mL), CXCL6 (1 μg/mL), Pleiotrophin (PTN)(1 μg/mL), SEMA6D (1 μg/mL), and TNF-α (1 ng/mL) were measured through electrical cell impendence sensing (ECIS) assay, and transwell assay, respectively. C) HLMVECs cultured on transwell treated with VEGF-D, followed by challenging with IL1β, TNF-α, or thrombin. The permeability was determined using a 20kDa FITC-dextran probe. D) Immunofluorescent images of F actin (red), and VE-Cadherin (green) in HLMVECs challenged with TNF-α or IL1β with or without VEGF-D. White boxes highlight VE-Cadherin junctions in all conditions. Scale bar 20 μm. E) Protein expression of phosphor-MLC in HLMVECs challenged with thrombin with different concentrations of VEGF-D was assessed through a western blot. Data presented as mean ± SEM. * and ** indicate p<0.05 and p<0.01, respectively.

Article Snippet: The following ligands were purchased from vendors: BMP5 (Novus Biologicals, # 615-BMC-020), VEGF-D (Cayman Chemical, #32055), Adrenomedullin (ADM)(Cayman Chemical, #24889), Angiopoietin-1 (Novus Biologicals, #923-AN), TNF-α (R&D Systems, # 10291-TA), Slit homolog 2 (SLIT2)(R&D Systems, # 8616-SL), CXCL6 (R&D Systems, # 333-GC-025/CF), Pleiotrophin (PTN)(R&D Systems, # 252-PL), and Semaphorin 6D (Sema6D)(R&D Systems, #2095-S6).

Techniques: Permeability, Transwell Assay, Cell Culture, Expressing, Western Blot

A Immunostaining of a murine knee joint at 18.5 dpc with an anti‐GCP‐2 or IgG control antibody. B Immunohistochemistry for GCP‐2 (red) in the knee of a human 5‐month old fetus. Scale bar = 100 μm. C, D Immunostaining for GCP‐2 in an adult (12‐week‐old) mouse knee (C) or in adult human articular cartilage (D). Scale bar = 50 μm in (C) and 100 μm in (D). Data information: E—epiphyseal cartilage; A—articular cartilage; M—meniscus; T—tibia; F—femur; T‐AC—tibial articular cartilage; F‐AC—femur articular cartilage; in (C), dotted lines indicate the osteochondral boundary and the cartilage surface and the arrow the bone marrow spaces. Source data are available online for this figure.

Journal: EMBO Molecular Medicine

Article Title: Disease modification and symptom relief in osteoarthritis using a mutated GCP ‐2/ CXCL6 chemokine

doi: 10.15252/emmm.202216218

Figure Lengend Snippet: A Immunostaining of a murine knee joint at 18.5 dpc with an anti‐GCP‐2 or IgG control antibody. B Immunohistochemistry for GCP‐2 (red) in the knee of a human 5‐month old fetus. Scale bar = 100 μm. C, D Immunostaining for GCP‐2 in an adult (12‐week‐old) mouse knee (C) or in adult human articular cartilage (D). Scale bar = 50 μm in (C) and 100 μm in (D). Data information: E—epiphyseal cartilage; A—articular cartilage; M—meniscus; T—tibia; F—femur; T‐AC—tibial articular cartilage; F‐AC—femur articular cartilage; in (C), dotted lines indicate the osteochondral boundary and the cartilage surface and the arrow the bone marrow spaces. Source data are available online for this figure.

Article Snippet: For loss‐of‐function experiments, densitometry quantification of Alcian blue was made for: HAC micromasses stimulated with 12.5 μg/ml GCP‐2 blocking antibody (Bio‐Techne MAB333) or IgG control (MAB002); C3H10T½ cells (ATCC CCL‐226) and C28/I2 cells were transfected with 25 nM of GCP‐2 siRNA (Thermofisher Silencer™ Select Pre‐Designed siRNA Catalog# 4392420) for 24 h and cultured in micromass. siRNA sequences: sense CUAUUGUAUUUCUAUCAUATT; antisense UAUGAUAGAAAUACAAUAGTT.

Techniques: Immunostaining, Immunohistochemistry

A–C GAG content as assessed by Alcian blue staining with densitometry (A) or spectrophotometric (B, C) quantitation. A HAC micromasses treated with a GCP‐2 neutralizing antibody ( n = 4) or IgG control ( n = 4); P ‐values were determined by the Mann–Whitney U ‐test. (B) C28/I2 micromasses treated with GCP‐2 siRNA ( n = 12) or scrambled control siRNA (Scr; n = 14); P ‐values were determined by unpaired two‐tailed Student's t ‐test. (C) C3H10T½ micromasses treated for 3 days with recombinant GCP‐2100 ng/ml ( n = 4) or vehicle ( n = 4) or BMP‐2 ( n = 4); P ‐values were determined by ANOVA followed by Tukey HSD post hoc test. D qPCR for ACAN (Aggrecan) expression in C3H10T½ micromasses treated for 3 days with recombinant GCP‐2 (100 ng/ml) ( n = 3) or vehicle ( n = 4); ACAN —Aggrecan; P ‐values were determined by unpaired two‐tailed Student's t ‐test. E Spectrophotometric quantification of GAG release (dimethylmethylene blue assay) in supernatant of human cartilage explants treated with recombinant GCP‐2 ( n = 5) or vehicle ( n = 5) and incubated for 24 h; P ‐values were determined by Mann–Whitney U ‐test. F Toluidine blue staining of ectopic cartilage explants collected 2 weeks after subcutaneous co‐implantation of HACs in a 10:1 ratio with growth‐arrested COS7 expressing either GFP ( n = 3) or GCP‐2 ( n = 3) and quantification—on the right‐hand side—of the metachromatically stained area as % of total area; scale bar = 200 μm; P ‐values were determined by unpaired two‐tailed Student's t ‐test. G Collagen 2 (Col2) immunostaining (left) and quantification (right) as % of total area; scale bar = 100 μm; n = 3 per group; P ‐values were determined by unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.

Journal: EMBO Molecular Medicine

Article Title: Disease modification and symptom relief in osteoarthritis using a mutated GCP ‐2/ CXCL6 chemokine

doi: 10.15252/emmm.202216218

Figure Lengend Snippet: A–C GAG content as assessed by Alcian blue staining with densitometry (A) or spectrophotometric (B, C) quantitation. A HAC micromasses treated with a GCP‐2 neutralizing antibody ( n = 4) or IgG control ( n = 4); P ‐values were determined by the Mann–Whitney U ‐test. (B) C28/I2 micromasses treated with GCP‐2 siRNA ( n = 12) or scrambled control siRNA (Scr; n = 14); P ‐values were determined by unpaired two‐tailed Student's t ‐test. (C) C3H10T½ micromasses treated for 3 days with recombinant GCP‐2100 ng/ml ( n = 4) or vehicle ( n = 4) or BMP‐2 ( n = 4); P ‐values were determined by ANOVA followed by Tukey HSD post hoc test. D qPCR for ACAN (Aggrecan) expression in C3H10T½ micromasses treated for 3 days with recombinant GCP‐2 (100 ng/ml) ( n = 3) or vehicle ( n = 4); ACAN —Aggrecan; P ‐values were determined by unpaired two‐tailed Student's t ‐test. E Spectrophotometric quantification of GAG release (dimethylmethylene blue assay) in supernatant of human cartilage explants treated with recombinant GCP‐2 ( n = 5) or vehicle ( n = 5) and incubated for 24 h; P ‐values were determined by Mann–Whitney U ‐test. F Toluidine blue staining of ectopic cartilage explants collected 2 weeks after subcutaneous co‐implantation of HACs in a 10:1 ratio with growth‐arrested COS7 expressing either GFP ( n = 3) or GCP‐2 ( n = 3) and quantification—on the right‐hand side—of the metachromatically stained area as % of total area; scale bar = 200 μm; P ‐values were determined by unpaired two‐tailed Student's t ‐test. G Collagen 2 (Col2) immunostaining (left) and quantification (right) as % of total area; scale bar = 100 μm; n = 3 per group; P ‐values were determined by unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.

Techniques: Staining, Quantitation Assay, MANN-WHITNEY, Two Tailed Test, Recombinant, Expressing, Dimethylmethylene Blue Assay, Incubation, Immunostaining

A, B C28/I2 (A) or C3H10T1/2 (B) monolayers transfected with 25 nM of either scrambled or GCP‐2 siRNA for 72 h and analyzed for GCP‐2 levels by Western blotting. (A) A representative western blot; (B) densitometric quantification of three independent experiments. N = 3. P ‐values were determined using the two‐tailed Student's t ‐test. C C28/I2 micromasses stimulated for 3 days with recombinant GCP‐2, IGF‐1 (positive control) or untreated (vehicle) and assessed for AKT phosphorylation levels by Western blotting; β‐actin as loading control; GCP‐2—WT, GCP‐2‐treated samples, IGF‐1—IGF‐1‐treated samples, V—vehicle‐treated samples, pAKT—phosphorylated AKT. D Alcian blue staining and spectrophotometric quantitation of GAGs in micromasses of HACs treated with recombinant GCP‐2 or vehicle ( n = 3); P ‐values were determined by unpaired, two‐tailed Student's t ‐test. OD—optical density. Source data are available online for this figure.

Journal: EMBO Molecular Medicine

Article Title: Disease modification and symptom relief in osteoarthritis using a mutated GCP ‐2/ CXCL6 chemokine

doi: 10.15252/emmm.202216218

Figure Lengend Snippet: A, B C28/I2 (A) or C3H10T1/2 (B) monolayers transfected with 25 nM of either scrambled or GCP‐2 siRNA for 72 h and analyzed for GCP‐2 levels by Western blotting. (A) A representative western blot; (B) densitometric quantification of three independent experiments. N = 3. P ‐values were determined using the two‐tailed Student's t ‐test. C C28/I2 micromasses stimulated for 3 days with recombinant GCP‐2, IGF‐1 (positive control) or untreated (vehicle) and assessed for AKT phosphorylation levels by Western blotting; β‐actin as loading control; GCP‐2—WT, GCP‐2‐treated samples, IGF‐1—IGF‐1‐treated samples, V—vehicle‐treated samples, pAKT—phosphorylated AKT. D Alcian blue staining and spectrophotometric quantitation of GAGs in micromasses of HACs treated with recombinant GCP‐2 or vehicle ( n = 3); P ‐values were determined by unpaired, two‐tailed Student's t ‐test. OD—optical density. Source data are available online for this figure.

Techniques: Transfection, Western Blot, Two Tailed Test, Recombinant, Positive Control, Staining, Quantitation Assay

A, B qPCR for (A) Runx2 and (B) Col1A1 expression in HAC micromasses treated for 24 h with recombinant GCP‐2 (100 ng/ml) or vehicle ( n = 4); Runx2 —Runt‐related transcription factor 2, Col1A1— collagen type 1 alpha 1; n = 4; P ‐values were determined by unpaired two‐tailed Student's t ‐test. C Alkaline phosphatase staining (red) in HAC monolayer after 3 week treatment with T3 hormone (100 ng/ml), T3 hormone + GCP‐2 (100 ng/ml), or control medium; scale bar 100 μm. D Quantification of alkaline phosphatase stained area ( n = 3); P ‐values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means. E qPCR for Col10A1 ( n = 3) of HAC 3 weeks after treatment with vehicle, T3 (100 ng/ml) or T3 + GCP‐2 (100 ng/ml); n = 3; P ‐values were determined by ANOVA with Tukey's HSD post hoc test. F–H C3H10T½ monolayers were treated with osteogenic medium (OM) or OM + GCP‐2 (100 ng/ml): (F) number of cells per well positive for alkaline phosphatase staining ( n = 3 in OM and n = 4 in OM + GCP‐2), (G) number of alkaline phosphatase positive nodules per well ( n = 7), and (H) spectrophotometric quantification of alizarin red staining at 570 nm ( n = 6); P ‐values were determined by unpaired, two‐tailed Student's t ‐test. Source data are available online for this figure.

Journal: EMBO Molecular Medicine

Article Title: Disease modification and symptom relief in osteoarthritis using a mutated GCP ‐2/ CXCL6 chemokine

doi: 10.15252/emmm.202216218

Figure Lengend Snippet: A, B qPCR for (A) Runx2 and (B) Col1A1 expression in HAC micromasses treated for 24 h with recombinant GCP‐2 (100 ng/ml) or vehicle ( n = 4); Runx2 —Runt‐related transcription factor 2, Col1A1— collagen type 1 alpha 1; n = 4; P ‐values were determined by unpaired two‐tailed Student's t ‐test. C Alkaline phosphatase staining (red) in HAC monolayer after 3 week treatment with T3 hormone (100 ng/ml), T3 hormone + GCP‐2 (100 ng/ml), or control medium; scale bar 100 μm. D Quantification of alkaline phosphatase stained area ( n = 3); P ‐values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means. E qPCR for Col10A1 ( n = 3) of HAC 3 weeks after treatment with vehicle, T3 (100 ng/ml) or T3 + GCP‐2 (100 ng/ml); n = 3; P ‐values were determined by ANOVA with Tukey's HSD post hoc test. F–H C3H10T½ monolayers were treated with osteogenic medium (OM) or OM + GCP‐2 (100 ng/ml): (F) number of cells per well positive for alkaline phosphatase staining ( n = 3 in OM and n = 4 in OM + GCP‐2), (G) number of alkaline phosphatase positive nodules per well ( n = 7), and (H) spectrophotometric quantification of alizarin red staining at 570 nm ( n = 6); P ‐values were determined by unpaired, two‐tailed Student's t ‐test. Source data are available online for this figure.

Techniques: Expressing, Recombinant, Two Tailed Test, Staining, Comparison

A Human GCP‐2 sequence aligned with closely related human CXCL proteins and the mouse CXCL5 (mCXCL5). The secondary structure is predicted based on the NMR structure of human CXCL5 (PDB: 2MGS). Conserved residues are highlighted in red boxes (white font) and partially conserved residues with similar chemical properties are shown in red font. B, C GAG binding capacity of wild‐type GCP‐2, and mutants (K101E single mutant, K105E single mutant, GCP‐2‐D = K101E_K105E double mutant and GCP‐2‐T = triple mutant K100E_ K101E_K105E) was assessed by (B) a heparin microtiter plate binding assay measuring the interaction of biotinylated‐heparin (b‐heparin) with immobilized proteins and (C) heparin affinity chromatography equilibrated and run in PBS, pH 7.4. In (C), the proteins are eluted with a salt gradient from 0 to 2 M NaCl monitored by the conductivity (Cond), where 20 and 90 mS/cm correspond to 193 and 1,360 mM NaCl, respectively; GCP‐2‐T, GCP‐2‐D, K101E, K105E and WT proteins elute at 530, 617, 815, 850 and 1,024 mM NaCl, respectively. D, E Maximum migratory activity of WT GCP‐2 (50 nM) was compared with GCP‐2 mutants (50 nM) in (D) chemotaxis and (E) transendothelial migration assays in CXCR2‐expressing 300‐19 pre‐B cells. WT—wild‐type GCP‐2; K101E—GCP‐2_K101E single mutant; K105E—GCP‐2_K105E single mutant; D—GCP‐2_K101E_K105E double mutant; T—GCP‐2_K100E_K101E_K105E triple mutant; n = 4; P ‐values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means. F–H C28/I2 micromasses stimulated with WT GCP‐2, GCP‐2 triple mutant (GCP‐2‐T) or untreated and assessed for (F) AKT phosphorylation levels by Western blotting; V—vehicle‐treated samples, GCP‐2—WT GCP‐2‐treated samples, GCP‐2‐T—triple mutant GCP‐2‐treated samples, pAKT—phosphorylated AKT, tAKT—total AKT. On the right, quantification of the density of the bands using ImageJ. Five independent experiments were analyzed. The data were scaled and P ‐values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means. (G) Spectrophotometric quantification of proteoglycan content by Alcian blue staining. P ‐values were determined by Kruskal–Wallis test with Dunn's post hoc test; ( n = 8). (H) ACAN expression levels by qPCR; ACAN —Aggrecan; P ‐values were determined by ANOVA with Tukey's HSD post hoc test; n = 4. I, J Real time quantitative PCR of COL2A1 (I) and COL10A1 (J) mRNA normalized to β actin. P ‐values were determined by fitting a generalized linear model followed by comparison of the estimated marginal means; n = 4 control, 5 GCP‐2 and GCP‐2‐T for COL2A1 and n = 6 per group for COL10A1. K Number of neutrophils counted within the intercondylar space on sections from mice killed 4 days after an intra‐articular injection of adenovirus encoding GFP ( n = 3), GCP‐2 ( n = 3) and GCP‐2‐T ( n = 3); P ‐values were determined by fitting a generalized linear model (family = poisson) followed by pairwise comparison of the estimated marginal means. Source data are available online for this figure.

Journal: EMBO Molecular Medicine

Article Title: Disease modification and symptom relief in osteoarthritis using a mutated GCP ‐2/ CXCL6 chemokine

doi: 10.15252/emmm.202216218

Figure Lengend Snippet: A Human GCP‐2 sequence aligned with closely related human CXCL proteins and the mouse CXCL5 (mCXCL5). The secondary structure is predicted based on the NMR structure of human CXCL5 (PDB: 2MGS). Conserved residues are highlighted in red boxes (white font) and partially conserved residues with similar chemical properties are shown in red font. B, C GAG binding capacity of wild‐type GCP‐2, and mutants (K101E single mutant, K105E single mutant, GCP‐2‐D = K101E_K105E double mutant and GCP‐2‐T = triple mutant K100E_ K101E_K105E) was assessed by (B) a heparin microtiter plate binding assay measuring the interaction of biotinylated‐heparin (b‐heparin) with immobilized proteins and (C) heparin affinity chromatography equilibrated and run in PBS, pH 7.4. In (C), the proteins are eluted with a salt gradient from 0 to 2 M NaCl monitored by the conductivity (Cond), where 20 and 90 mS/cm correspond to 193 and 1,360 mM NaCl, respectively; GCP‐2‐T, GCP‐2‐D, K101E, K105E and WT proteins elute at 530, 617, 815, 850 and 1,024 mM NaCl, respectively. D, E Maximum migratory activity of WT GCP‐2 (50 nM) was compared with GCP‐2 mutants (50 nM) in (D) chemotaxis and (E) transendothelial migration assays in CXCR2‐expressing 300‐19 pre‐B cells. WT—wild‐type GCP‐2; K101E—GCP‐2_K101E single mutant; K105E—GCP‐2_K105E single mutant; D—GCP‐2_K101E_K105E double mutant; T—GCP‐2_K100E_K101E_K105E triple mutant; n = 4; P ‐values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means. F–H C28/I2 micromasses stimulated with WT GCP‐2, GCP‐2 triple mutant (GCP‐2‐T) or untreated and assessed for (F) AKT phosphorylation levels by Western blotting; V—vehicle‐treated samples, GCP‐2—WT GCP‐2‐treated samples, GCP‐2‐T—triple mutant GCP‐2‐treated samples, pAKT—phosphorylated AKT, tAKT—total AKT. On the right, quantification of the density of the bands using ImageJ. Five independent experiments were analyzed. The data were scaled and P ‐values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means. (G) Spectrophotometric quantification of proteoglycan content by Alcian blue staining. P ‐values were determined by Kruskal–Wallis test with Dunn's post hoc test; ( n = 8). (H) ACAN expression levels by qPCR; ACAN —Aggrecan; P ‐values were determined by ANOVA with Tukey's HSD post hoc test; n = 4. I, J Real time quantitative PCR of COL2A1 (I) and COL10A1 (J) mRNA normalized to β actin. P ‐values were determined by fitting a generalized linear model followed by comparison of the estimated marginal means; n = 4 control, 5 GCP‐2 and GCP‐2‐T for COL2A1 and n = 6 per group for COL10A1. K Number of neutrophils counted within the intercondylar space on sections from mice killed 4 days after an intra‐articular injection of adenovirus encoding GFP ( n = 3), GCP‐2 ( n = 3) and GCP‐2‐T ( n = 3); P ‐values were determined by fitting a generalized linear model (family = poisson) followed by pairwise comparison of the estimated marginal means. Source data are available online for this figure.

Techniques: Sequencing, Binding Assay, Mutagenesis, Affinity Chromatography, Activity Assay, Chemotaxis Assay, Migration, Expressing, Comparison, Western Blot, Staining, Real-time Polymerase Chain Reaction, Injection

A 3D homology model of human GCP‐2 based on the NMR structure of human CXCL5 dimer (PDB: 2MGS) generated using SWISS‐MODEL. The GCP‐2 monomers are colored blue and green with Lys100, Lys101 and Lys105 shown in space filling representation for the latter. B Comparison of 1D NMR spectra of WT and mutant GCP‐2 proteins (all human) made in this study. C SDS–PAGE analysis of GCP‐2 WT and mutants under reducing (R) and nonreducing (NR) conditions. Protein samples (1 μg) were either reduced and alkylated (R) or alkylated (NR). MW size from protein markers is displayed on the left. WT—wild‐type GCP‐2; K101E—K101E GCP‐2_single mutant; K105E—K105E GCP‐2_single mutant; D—K101E_K105E GCP‐2_double mutant; T—K100E_K101E_K105E GCP‐2_triple mutant. D, E Dose‐dependency of WT GCP‐2‐induced (D) chemotaxis and (E) and transendothelial migration (TEM) of CXCR2‐expressing 300‐19 pre‐B cell line (mean values ± SEM). N = 4. F Representative images of immunostaining for the Ly6G neutrophil marker in the intercondylar notch of mice injected intra‐articularly with GFP, GCP‐2, or GCP‐2‐T adenovirus. Time point: 2 days. White arrowheads indicate neutrophils. Scale bar—50 μm. F—femur; CL—cruciate ligament. G Average thickness of the synovial membrane 4 days after the intra‐articular injection of adenovirus encoding GFP, GCP‐2 or GCP‐2‐T. The thickness of the synovial membrane was assessed by histomorphometry using ImageJ. P ‐values were determined with ANOVA n = 4 per group. H Caliper measurement of knee size 4 days after the injection of GFP, GCP‐2 and GCP‐2‐T adenovirus; P ‐values were determined by one‐way ANOVA ( n = 4). Source data are available online for this figure.

Journal: EMBO Molecular Medicine

Article Title: Disease modification and symptom relief in osteoarthritis using a mutated GCP ‐2/ CXCL6 chemokine

doi: 10.15252/emmm.202216218

Figure Lengend Snippet: A 3D homology model of human GCP‐2 based on the NMR structure of human CXCL5 dimer (PDB: 2MGS) generated using SWISS‐MODEL. The GCP‐2 monomers are colored blue and green with Lys100, Lys101 and Lys105 shown in space filling representation for the latter. B Comparison of 1D NMR spectra of WT and mutant GCP‐2 proteins (all human) made in this study. C SDS–PAGE analysis of GCP‐2 WT and mutants under reducing (R) and nonreducing (NR) conditions. Protein samples (1 μg) were either reduced and alkylated (R) or alkylated (NR). MW size from protein markers is displayed on the left. WT—wild‐type GCP‐2; K101E—K101E GCP‐2_single mutant; K105E—K105E GCP‐2_single mutant; D—K101E_K105E GCP‐2_double mutant; T—K100E_K101E_K105E GCP‐2_triple mutant. D, E Dose‐dependency of WT GCP‐2‐induced (D) chemotaxis and (E) and transendothelial migration (TEM) of CXCR2‐expressing 300‐19 pre‐B cell line (mean values ± SEM). N = 4. F Representative images of immunostaining for the Ly6G neutrophil marker in the intercondylar notch of mice injected intra‐articularly with GFP, GCP‐2, or GCP‐2‐T adenovirus. Time point: 2 days. White arrowheads indicate neutrophils. Scale bar—50 μm. F—femur; CL—cruciate ligament. G Average thickness of the synovial membrane 4 days after the intra‐articular injection of adenovirus encoding GFP, GCP‐2 or GCP‐2‐T. The thickness of the synovial membrane was assessed by histomorphometry using ImageJ. P ‐values were determined with ANOVA n = 4 per group. H Caliper measurement of knee size 4 days after the injection of GFP, GCP‐2 and GCP‐2‐T adenovirus; P ‐values were determined by one‐way ANOVA ( n = 4). Source data are available online for this figure.

Techniques: Generated, Comparison, Mutagenesis, SDS Page, Chemotaxis Assay, Migration, Expressing, Immunostaining, Marker, Injection, Membrane

A Ten‐week‐old female mice were injected intra‐articularly with 6 μl of GFP ( n = 4), GCP‐2 ( n = 3) or GCP‐2‐T ( n = 3) adenovirus and killed 4 days later for immunofluorescence analysis of phospho‐AKT (pAKT). After thresholding, pAKT + cells were counted using ImageJ. P ‐values were obtained by fitting a generalized linear model (family = Poisson) followed by pairwise comparison of the estimated marginal means. B–E Sections from the MLI experiment in Fig were used to assess the expression of (B) collagen type II (Col2) ( n = 4 for GFP and 3 for GCP‐2 and GCP‐2‐T), (C) collagen type X (Col10) ( n = 3 for GFP and 4 for GCP‐2 and GCP‐2‐T), (D) NITEGE neo‐epitope ( n = 6 for GFP, n = 2 for GCP‐2 and n = 4 for GCP‐2‐T) using immunofluorescence, and (E) apoptosis using the TUNEL assay ( n = 3 for GFP and n = 4 for GCP‐2‐T); P ‐values were obtained by fitting a generalized linear model. Whenever multiple technical replicates from the same knee were available, individual values were averaged. Scale bar = 50 μm. Source data are available online for this figure.

Journal: EMBO Molecular Medicine

Article Title: Disease modification and symptom relief in osteoarthritis using a mutated GCP ‐2/ CXCL6 chemokine

doi: 10.15252/emmm.202216218

Figure Lengend Snippet: A Ten‐week‐old female mice were injected intra‐articularly with 6 μl of GFP ( n = 4), GCP‐2 ( n = 3) or GCP‐2‐T ( n = 3) adenovirus and killed 4 days later for immunofluorescence analysis of phospho‐AKT (pAKT). After thresholding, pAKT + cells were counted using ImageJ. P ‐values were obtained by fitting a generalized linear model (family = Poisson) followed by pairwise comparison of the estimated marginal means. B–E Sections from the MLI experiment in Fig were used to assess the expression of (B) collagen type II (Col2) ( n = 4 for GFP and 3 for GCP‐2 and GCP‐2‐T), (C) collagen type X (Col10) ( n = 3 for GFP and 4 for GCP‐2 and GCP‐2‐T), (D) NITEGE neo‐epitope ( n = 6 for GFP, n = 2 for GCP‐2 and n = 4 for GCP‐2‐T) using immunofluorescence, and (E) apoptosis using the TUNEL assay ( n = 3 for GFP and n = 4 for GCP‐2‐T); P ‐values were obtained by fitting a generalized linear model. Whenever multiple technical replicates from the same knee were available, individual values were averaged. Scale bar = 50 μm. Source data are available online for this figure.

Techniques: Injection, Immunofluorescence, Comparison, Expressing, TUNEL Assay

A Schematic of in vivo experimental osteoarthritis. B Weekly pain measurement by incapacitance, shown as the percentage of bodyweight loaded on the operated leg in GFP ( n = 15) versus GCP‐2 ( n = 14) and GFP versus GCP‐2‐T ( n = 15) treated mice. Circles show the mean, and error bars show 95% confidence intervals. P ‐values were determined by building a mixed‐effect linear model. * P < 0.05. C Incapacitance at Week 10 (final time point); red line indicates 50% loading (no pain); P ‐values were determined by unpaired, two‐tailed one sample Student's t ‐test testing the hypothesis that mice loaded 50% body weight on the operated limb ( n = 13 in the GFP group and n = 14 in the GCP‐2 and GCP‐2‐T groups). D The area under the curve (AUC) of incapacitance calculated starting from Week 6; P ‐values were determined by ANOVA with Tukey's HSD post hoc test ( n = 13 in the GFP group and 14 in the GCP‐2 and GCP‐2‐T group). E, F Osteoarthritis severity assessed using (E) OARSI scoring system 10 weeks after MLI; GFP ( n = 7), GCP‐2 ( n = 8) and GCP‐2‐T ( n = 7); P ‐values were determined by fitting a generalized linear model followed by comparison of the estimated marginal means. F representative image (Safranin O staining) for each treatment; arrows indicate the tidemark; scale bar 100 μm. G Quantification of osteophytes area; GFP ( n = 7), GCP‐2 ( n = 7) and GCP‐2‐T ( n = 8); P ‐values were determined by ANOVA with Tukey's HSD post hoc test. H Representative images with osteophyte highlighted; scale bar 300 μm. I Osteophyte maturity score ( n = 7); P ‐values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means. Source data are available online for this figure.

Journal: EMBO Molecular Medicine

Article Title: Disease modification and symptom relief in osteoarthritis using a mutated GCP ‐2/ CXCL6 chemokine

doi: 10.15252/emmm.202216218

Figure Lengend Snippet: A Schematic of in vivo experimental osteoarthritis. B Weekly pain measurement by incapacitance, shown as the percentage of bodyweight loaded on the operated leg in GFP ( n = 15) versus GCP‐2 ( n = 14) and GFP versus GCP‐2‐T ( n = 15) treated mice. Circles show the mean, and error bars show 95% confidence intervals. P ‐values were determined by building a mixed‐effect linear model. * P < 0.05. C Incapacitance at Week 10 (final time point); red line indicates 50% loading (no pain); P ‐values were determined by unpaired, two‐tailed one sample Student's t ‐test testing the hypothesis that mice loaded 50% body weight on the operated limb ( n = 13 in the GFP group and n = 14 in the GCP‐2 and GCP‐2‐T groups). D The area under the curve (AUC) of incapacitance calculated starting from Week 6; P ‐values were determined by ANOVA with Tukey's HSD post hoc test ( n = 13 in the GFP group and 14 in the GCP‐2 and GCP‐2‐T group). E, F Osteoarthritis severity assessed using (E) OARSI scoring system 10 weeks after MLI; GFP ( n = 7), GCP‐2 ( n = 8) and GCP‐2‐T ( n = 7); P ‐values were determined by fitting a generalized linear model followed by comparison of the estimated marginal means. F representative image (Safranin O staining) for each treatment; arrows indicate the tidemark; scale bar 100 μm. G Quantification of osteophytes area; GFP ( n = 7), GCP‐2 ( n = 7) and GCP‐2‐T ( n = 8); P ‐values were determined by ANOVA with Tukey's HSD post hoc test. H Representative images with osteophyte highlighted; scale bar 300 μm. I Osteophyte maturity score ( n = 7); P ‐values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means. Source data are available online for this figure.

Techniques: In Vivo, Two Tailed Test, Comparison, Staining

Bone density of subchondral bone in operated and sham‐operated knees as accessed by microCT as BV/TV; P ‐values were determined by fitting a mixed‐effect model followed by pairwise comparison of the estimated marginal means ( n = 14 for the GFP and GCP‐2 group and 12 for GCP‐2‐T). Density of subchondral bone in different treatments as accessed by microCT as BV/TV; GFP ( n = 7), GCP‐2 ( n = 7) and GCP‐2‐T ( n = 6); P ‐values were determined by ANOVA with Tukey's HSD post hoc test. Synovium thickness in μm assessed as average thickness of synovium in six areas of the joint; GFP ( n = 7), GCP‐2 ( n = 7) and GCP‐2‐T ( n = 7); P ‐values were determined by ANOVA with Tukey's HSD post hoc test. Source data are available online for this figure.

Journal: EMBO Molecular Medicine

Article Title: Disease modification and symptom relief in osteoarthritis using a mutated GCP ‐2/ CXCL6 chemokine

doi: 10.15252/emmm.202216218

Figure Lengend Snippet: Bone density of subchondral bone in operated and sham‐operated knees as accessed by microCT as BV/TV; P ‐values were determined by fitting a mixed‐effect model followed by pairwise comparison of the estimated marginal means ( n = 14 for the GFP and GCP‐2 group and 12 for GCP‐2‐T). Density of subchondral bone in different treatments as accessed by microCT as BV/TV; GFP ( n = 7), GCP‐2 ( n = 7) and GCP‐2‐T ( n = 6); P ‐values were determined by ANOVA with Tukey's HSD post hoc test. Synovium thickness in μm assessed as average thickness of synovium in six areas of the joint; GFP ( n = 7), GCP‐2 ( n = 7) and GCP‐2‐T ( n = 7); P ‐values were determined by ANOVA with Tukey's HSD post hoc test. Source data are available online for this figure.

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