wnt5a neutralizing antibody  (R&D Systems)

Journal: BMB Reports

Article Title: Wnt5a exacerbates pathological bone features and trabecular bone loss in curdlan-injected SKG mice via osteoclast activation

doi: 10.5483/BMBRep.2024-0155

Figure Lengend Snippet: Treatment with WNT5A aggravates inflammation and pathological bone features in curdlan-injected SKG mice in vivo . Curdlan-administered SKG mice were intravenously injected with either vehicle or 100 μg of recombinant Wnt5a and observed for up to 5 weeks (n = 5 mice per group). (A) Experimental design. (B and C) Clinical arthritis scores and hindpaw thickness were measured. (D) Representative gross images (upper) and microCT images (lower) of each group, with orange coloration indicating inflamed bone in the hindpaw. (E) Representative images taken at 5 weeks, including microCT (upper), H&E and safranin O staining (middle), and TRAP staining (lower) in the ankle. White and black arrows indicate ectopic bone formation in the tibia. (F) Histopathological assessment of the ankle was performed using a scoring method for each group. H&E, Hematoxylin & Eosin; SO, Safranin O; EB, Ectopic bone; T, Tibia. Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01 by Mann–Whitney U test.

Article Snippet: Human/mouse recombinant Wnt5a (645-WN; R&D Systems) and anti-Wnt5a (MAB645; R&D Systems) were used for stimulation and neutralization, respectively.

Techniques: Injection, In Vivo, Recombinant, Staining, MANN-WHITNEY

Journal: BMB Reports

Article Title: Wnt5a exacerbates pathological bone features and trabecular bone loss in curdlan-injected SKG mice via osteoclast activation

doi: 10.5483/BMBRep.2024-0155

Figure Lengend Snippet: Treatment with Wnt5a facilitated an increase in osteoclast activity and trabecular bone loss of curdlan-injected SKG mice in vivo . The Vehicle and Wnt5a-treated groups were analyzed using the following: (A) representative microCT images of femurs from each group; (B) quantitative microCT data, including trabecular volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation; (C) ELISA measurements of PINP (bone formation marker) and CTX-1 (bone resorption marker); and (D) representative H&E and TRAP staining images of femurs, alongside quantification of TRAP-positive areas. Osteoclast differentiation of bone marrow–derived OCPs from SKG mice was assessed via (E) TRAP and F-actin staining and (F) osteoclast-differentiation markers like Itgb3, Dcstamp, Nfatc1, Ctsk, and Acp5 . Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01 by Mann–Whitney U test.

Article Snippet: Human/mouse recombinant Wnt5a (645-WN; R&D Systems) and anti-Wnt5a (MAB645; R&D Systems) were used for stimulation and neutralization, respectively.

Techniques: Activity Assay, Injection, In Vivo, Enzyme-linked Immunosorbent Assay, Marker, Staining, Derivative Assay, MANN-WHITNEY

Journal: BMB Reports

Article Title: Wnt5a exacerbates pathological bone features and trabecular bone loss in curdlan-injected SKG mice via osteoclast activation

doi: 10.5483/BMBRep.2024-0155

Figure Lengend Snippet: Wnt5a regulates osteoclast differentiation of OCPs through activation of the Rank/p38/Nfatc1 in vitro . OCPs from SKG mice were differentiated with IgG or anti-WNT5A in the presence or absence of Wnt5a. At 4 days, the cells were analyzed by (A) TRAP and F-actin staining (left) and quantification of TRAP-positive cells per well (right). (B) RT-qPCR for osteoclast-differentiation markers, including Itgb3, Dcstamp, Nfatc1, Ctsk , and Acp5 , and (C) immunoblotting for concentrations of osteoclast-related Rank, phos-p38, total-p38, and Nfatc1, were also performed. (D) Immunoblotting images were quantified using Image J. Values are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 by One-Way ANOVA.

Article Snippet: Human/mouse recombinant Wnt5a (645-WN; R&D Systems) and anti-Wnt5a (MAB645; R&D Systems) were used for stimulation and neutralization, respectively.

Techniques: Activation Assay, In Vitro, Staining, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Science advances

Article Title: A fibronectin gradient remodels mixed-phase mesoderm.

doi: 10.1126/sciadv.adl6366

Figure Lengend Snippet: Fig. 5. Feedback mechanism involving Wnt5a-YAP-FN. (A) Transverse sections of 21 som. CD1 embryos at forelimb anterior and posterior regions. Sections were stained with 4′,6-diamidino-2-phenylindole (DAPI; cyan) anti-YAP (green) and anti-fibronectin antibody (red). (B) Relative fibronectin fluorescence intensity versus YAP nuclear/ cytoplasmic ratio of 20 to 21 som. CD1 (n = 3) forelimbs. PCC, Pearson correlation coefficient. (C) YAP nuclear/cytoplasmic ratio of 20 to 21 som. CD1 embryos (n = 3) at forelimb anterior and posterior regions (two-tailed unpaired Student’s t test, ****P < 0.0001). (D) Transverse sections of 20 som. T:Cre;Yapf/f;Tazf/+ embryos at forelimb anterior and posterior regions. Sections were stained with DAPI (cyan) anti-YAP (green) and anti-fibronectin antibody (red). (E) Relative fibronectin fluorescence intensity in 20 to 21 som. Yapf/+;Tazf/+ (n = 5) versus T:Cre;Yapf/f;Tazf/+ (n = 3) embryos at forelimb anterior and posterior regions (two-tailed unpaired Student’s t test, **P < 0.01). Error bars indicate SD. (F) Limb bud shape change among T:Cre;Yapf/f;Tazf/+ embryos from 20 to 25 som. stage reconstructed from OPT. (G) Schematic model representing the WNT5A-YAP-FN signaling pathway that establishes tissue stiffness and orchestrates cell movements to drive limb bud shape change. (H) Variations in stiffness gradient geometry correspond to patterns of morphogenetic cell movements in the limb bud (top) and mandibular arch (bottom).

Article Snippet: Primary antibodies included fibronectin (1:100; Abcam, lot ab2413), pHH3 (1:250; Cell Signaling Technology, lot 9706), N- cadherin (1:200; BD Biosciences, lot 610920), β- catenin (1:200; Abcam, lot ab59430), Vangl2 (1:200; Sigma- Aldrich, lot HPA027043), p- SAPK/JNK (1:200; Cell Signaling Technology, lot 9255), YAP (1:200; Abcam, lot ab56701), FAK (1:200; BD Biosciences, lot 610087), phospho- YAP (1:200; Cell Signaling Technology, lot 4911), and WNT5A (1:200; R&D Systems, lot MAB645).

Techniques: Staining, Fluorescence, Two Tailed Test