recombinant mouse sfrp1 protein  (R&D Systems)

Journal: JVS-Vascular Science

Article Title: Vascular adhesion molecule 1 + fibro-adipogenic progenitors mark fatty infiltration in chronic limb-threatening ischemia

doi: 10.1016/j.jvssci.2025.100295

Figure Lengend Snippet: Sfrp1 regulates Vcam1 + fibro-adipogenic progenitor (FAP) adipogenic differentiation. (A) Dot plot demonstrating Wnt signaling pathways in Vcam1 + vs Vcam1 − FAPs. (B) Bar graph demonstrating Sfrp1 expression in Vcam1 + vs Vcam1 − FAPs. (C) Heatmap representing Pearson's correlation values of Sfrp1 and Vcam1 expression in FAPs. (D) Representative images of Oil Red O (ORO) staining of Vcam1 + FAPs treated with small interfering RNA ( siRNA ) against Sfrp1 and a control siRNA and quantification of ORO staining; data shown as mean ± standard error of the mean, ∗∗∗ P < .001. (E) Representative images of perilipin ( green ) and DAPI ( blue ) of Vcam1 + FAPs treated with siRNA against Sfrp1 and a control siRNA and quantification of perilipin staining; data shown as mean ± standard error of the mean, ∗ P < .05 (F) Representative images of peroxisome proliferator-activated receptor ( PPAR-γ ) ( green ) and DAPI ( blue ) of Vcam1 + FAPs treated with siRNA against Sfrp1 and a control siRNA and quantification of PPAR-γ staining; data shown as mean ± standard error of the mean, ∗∗∗ P < .001. n = 3 experimental replicates for D - F . OD , optical density.

Article Snippet: FACS-sorted Vcam1 − FAPs were grown to 60% to 80% confluence followed by adipogenic differentiation and were treated with 500 ng/mL recombinant mouse Sfrp1 protein (R&D systems, 9019-SF) with or without sFRP-1 inhibitor, 2 μmol/L Way-316606 hydrochloride (Tocris, 4767), or vehicle control (DMSO).

Techniques: Protein-Protein interactions, Expressing, Staining, Small Interfering RNA, Control

Journal: JVS-Vascular Science

Article Title: Vascular adhesion molecule 1 + fibro-adipogenic progenitors mark fatty infiltration in chronic limb-threatening ischemia

doi: 10.1016/j.jvssci.2025.100295

Figure Lengend Snippet: Single cell RNA sequencing (RNA-seq) and single cell ATAC sequencing identifies Nr3c1 as a transcription factor (TF) that regulates fibro-adipogenic progenitor (FAP) adipogenesis. (A) Inferred TFs that regulate differential genes of Vcam1 + vs Vcam1 − FAPs. (B) Enhancers with regulation potential to Sfrp1 ( left ) and TF binding analysis to the enhancers. (C) Nr3c1 protein expression in Vcam1 + and Vcam1 − FAPs in adipogenic media for 3 days (n = technical replicates). (D) Representative images and quantification of Oil Red O (ORO) staining in Nr3c1-silenced Vcam1 + FAPs. Data shown as mean ± standard error of the mean, ∗∗ P < .01. (E and F) Representative images and quantification of perilipin ( green ), peroxisome proliferator-activated receptor gamma ( PPAR-γ ) ( green ), and DAPI ( blue ) staining in Nr3c1-silenced Vcam1 + FAPs. Data shown as mean ± standard error of the mean, ∗ P < .05, ∗∗∗ P < .001. n = 3 replicates for all experimental groups (C-F) . OD , optical density.

Article Snippet: FACS-sorted Vcam1 − FAPs were grown to 60% to 80% confluence followed by adipogenic differentiation and were treated with 500 ng/mL recombinant mouse Sfrp1 protein (R&D systems, 9019-SF) with or without sFRP-1 inhibitor, 2 μmol/L Way-316606 hydrochloride (Tocris, 4767), or vehicle control (DMSO).

Techniques: RNA Sequencing, Sequencing, Binding Assay, Expressing, Staining

Journal: JVS-Vascular Science

Article Title: Vascular adhesion molecule 1 + fibro-adipogenic progenitors mark fatty infiltration in chronic limb-threatening ischemia

doi: 10.1016/j.jvssci.2025.100295

Figure Lengend Snippet: Human chronic limb-threatening ischemia ( CLTI ) fibro-adipogenic progenitors ( FAPs ) display a myosteatosis transcriptional signature. (A) Uniform manifold approximation projection ( UMAP ) of FAPs in human PAD dataset, color represents subcluster. (B) UMAP of FAPs in human peripheral arterial disease ( PAD ) dataset, color represents location in limb. (C-E) Violin plot of Vcam1, Sfrp1, and Nr3c1 expression in distal/ischemic vs proximal/nonischemic FAPs. (F) Schematic summarizing study findings. Figures generated with Biorender.com .

Article Snippet: FACS-sorted Vcam1 − FAPs were grown to 60% to 80% confluence followed by adipogenic differentiation and were treated with 500 ng/mL recombinant mouse Sfrp1 protein (R&D systems, 9019-SF) with or without sFRP-1 inhibitor, 2 μmol/L Way-316606 hydrochloride (Tocris, 4767), or vehicle control (DMSO).

Techniques: Expressing, Generated

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: sFRP1 was abundantly produced in nerve ECM following injury and associated with nerve degeneration (A) The isolation of sciatic nerve samples and proteomic analysis process. (B) The clustering distribution of injured and uninjured nerve samples as plotted by PCA analysis. (C) Differentially expressed proteins between injured and uninjured nerve samples are displayed in volcano plot. N = 3 mice. Proteins regulated over 1.5-fold changes (adj. p < 0.05) are highlighted in blue (downregulated) and red (upregulated). (D) GO enrichment analysis indicating the classification of differentially expressed proteins related to the biological process category. (E) Differentially expressed proteins in the GO category of ECM are displayed as a heatmap. (F) Western blotting analysis demonstrating increased production of sFRP1 in the injured nerve tissue. (G) Quantification of sFRP1 protein level in sciatic nerves isolated from uninjured and injured mice as indicated by western blot analysis. N = 3 mice. (H) Representative TEM, HE, and TB images of injured nerves isolated from mice treated with WAY-316606 and PBS at 3 weeks post injury. N = 6 mice. (I and J) Quantification of myelinated axon diameter and g-ratio as indicated in TEM images. (K) Quantification of myelinated axon density as indicated in HE-stained images. (L) Representative IHC images of human nerves stained for sFRP1 at 12 h after injury. Statistical significance was determined using two-tailed unpaired Student’s t tests; ∗∗ p < 0.01; ∗ p < 0.05; ns, no difference. Data were presented as mean ± SD.

Article Snippet: Recombinant sFRP1 protein , MedChemExpress , Cat#HY-P73413.

Techniques: Produced, Isolation, Western Blot, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: SCs sensed injury signals to release sFRP1 and elicited mesenchymal traits (A) t-distributed stochastic neighbor embedding (t-SNE) plot shows clustering of nerve cells based on gene expression. Single-cell sequencing datasets are analyzed from GSE120678 . BC, B cell; TC, T cell; EC, endothelial cell; Macro, macrophage; SC, Schwann cell; Endo, endoneurial fibroblast; Epi, epineurial fibroblast; Peri, perineurial fibroblast. (B) sFRP1 expression is mainly distributed in SCs and fibroblasts in both uninjured and injured sciatic nerves. (C and D) Double IF staining of S100β (red)/sFRP1 (green) and Fibro (red)/sFRP1 (green) on both longitudinal (C) and transverse (D) sections of sciatic nerves. (E and F) Percentage of sFRP1-positive SCs and fibroblasts in uninjured and injured nerves. N = 6 mice. (G) Illustration of the in vivo LPS treatment design. (H) sFRP1 protein level in SCs isolated from PBS or LPS (15 mg/kg) intraperitoneally treated mice. (I) Quantification of sFRP1 protein level in sciatic nerves isolated from LPS-treated and PBS-treated mice as indicated by western blot analysis. N = 3 biological replicates. (J) Illustration of the in vitro LPS treatment design. (K) Western blot analysis of sFRP1 protein level in SCs treated with different concentrations of LPS. (L and M) Quantification of fluorescence intensity of sFRP1 and PDGFRα staining in SCs. (N) sFRP1 (green) and PDGFRα (red) double staining on LPS-treated and PBS-treated SCs. N = 3 biological replicates. Two fields were quantified as technical replicates in each biological replicates. Statistical significance was determined using two-tailed unpaired Student’s t tests; ∗∗∗∗ p < 0.0001; ∗∗ p < 0.01; ∗ p < 0.05. Data were presented as mean ± SD.

Article Snippet: Recombinant sFRP1 protein , MedChemExpress , Cat#HY-P73413.

Techniques: Gene Expression, Single Cell, Sequencing, Expressing, Staining, In Vivo, Isolation, Western Blot, In Vitro, Fluorescence, Double Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Mice with deletion of sFRP1 in SCs profoundly reduced macrophage infiltration and improved nerve regeneration (A) Sfrp1 flox/flox mice were bred with Plpcre Ert1 mice to generate tamoxifen-inducible SC-specific sFRP1 knockout ( Sfrp1 flox/flox Plpcre Ert1 ) and littermate control ( Sfrp1 flox/flox ) mice. (B and C) Representative SCG10 immunostaining and related quantification of sciatic nerves at 14 days post transection. N = 6 mice. The dashed line indicates the transection site. Scale bar, 500 μm. (D and E) Representative F4/80 immunostaining (red) of sciatic nerves taken from the injury site, 1,000, 2,000, and 3,000 μm distal to the injury site and related quantification of infiltrated macrophages. Scale bar, 100 μm. N = 6 mice. (F and G) Western blot analysis and related quantification of TNF-α level in injured nerves at 24 h post transection. N = 3 mice. (H and I) Triple staining of CCL2 (green), F4/80 (red), and NeuN (pink) on sciatic DRG sections from Sfrp1 flox/flox and Sfrp1 flox/flox Plpcre Ert1 mice and related quantification of CCL expression level in DRGs. N = 6 mice. No significant difference of CCL2 expression is observed between DRGs of Sfrp1 flox/flox and Sfrp1 flox/flox Plpcre Ert1 mice. (J–L) Representative TUBB3 immunostaining (green) of sciatic DRG neurons isolated from Sfrp1 flox/flox and Sfrp1 flox/flox Plpcre Ert1 mice ( n = 6 mice) and related quantification. DRG neurons were cultured in vitro for 4 days or 7 days. (M and N) Representative immunostaining and related quantification of ATF3 (red) and the neuronal marker NeuN (green) in sciatic DRGs at 24 h after nerve injury. N = 6 mice. Scale bar, 50 μm. Statistical significance in (C) and (E) was analyzed by two-way ANOVA followed by Sidak’s post hoc analysis. Statistical significance was determined using two-tailed unpaired Student’s t tests; ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns, no significance. Data were presented as mean ± SD.

Article Snippet: Recombinant sFRP1 protein , MedChemExpress , Cat#HY-P73413.

Techniques: Knock-Out, Control, Immunostaining, Western Blot, Staining, Expressing, Isolation, Cell Culture, In Vitro, Marker, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: SFRP1 induces the F4/80 + CD86 + proinflammatory macrophage phenotype and inhibits oxidative metabolism (A and B) The axon length of sciatic DRG neurons demonstrates no significant difference in response to sFRP1 treatment. N = 6 biological replicates. (C) Representative TEM images reveal that the morphology and structure of mitochondria were well preserved in sFRP1-treated neurons. (D and E) Representative TEM images and related quantification of nerve transections ( N = 6 mice). The suppressing effect of sFRP1 on axon regrowth is alleviated in a macrophage-deficient condition. (F) Double staining of IL-1β (red) and TNF-α (green) on sFRP1-treated BMDMs. (G) sFRP1-induced phenotypic switch is revealed by flow cytometric quantification. FITC reflects F4/80-positive cells. PE reflects CD206-positive cells. APC reflects CD86-positive cells. (H and I) Quantification of the percentage of IL-1β and TNF-α-positive cells as reflected by Figure 4 F. Biological replicates n = 3 with two technical replicates each. (J) Double staining of Arg-1 (red) and Wnt3a (green) on sFRP1 and PBS-treated BMDMs. (K) The internalizing capacity of BMDMs was measured by incubating with 100 μg/mL pHrodo BioParticles (green). BMDMs were visualized by F4/80 (red) staining. (L and M) Quantification of the percentage of proinflammatory and pro-resolving macrophages as reflected by Figure 4 G. Biological replicates n = 3 with two technical replicates each. (N) Heatmap of differentially expressed genes between sFRP1-treated and PBS-treated macrophages (control) based on RNA sequencing. N = 3 biological replicates. (O) GO classification of differentially expressed genes related to the biological process, cellular component, and molecular function categories. (P) KEGG enrichment analysis of differentially expressed genes based on RNA sequencing. (Q) Schematic diagram of the detection of mitochondrial respiration and glycolysis of macrophages by measuring OCR and ECAR. (R) OCR of macrophages at baseline and after serial administration with oligomycin, FCCP, and rotenone plus Antimycin A. Macrophages were treated with 50, 100, 200, and 500 nM sFRP1. (S) ECAR was compared at baseline and after serial administration with glucose, oligomycin, and 2-DG. Statistical significance in (E), (H), and (I) was determined using one-way ANOVA followed by Tukey’s multiple comparisons tests; ∗∗∗∗ p < 0.0001 versus PBS group; ∗∗ p < 0.01 versus PBS group. Statistical significance in (B), (L), and (M) was determined using two-tailed unpaired Student’s t tests; ∗∗∗ p < 0.001 versus PBS group; ns, no significance. Data were presented as mean ± SD.

Article Snippet: Recombinant sFRP1 protein , MedChemExpress , Cat#HY-P73413.

Techniques: Double Staining, Staining, Control, RNA Sequencing, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Identification of HSP90 as a binding protein to mediate the proinflammatory effect of sFRP1 on BMDMs (A) List of candidates with top 10 scores in LC-MS/MS analysis of BMDM-derived proteins with incubation of His-labeled sFRP1. (B) IP-MS analysis identifies HSP90 as an interacting protein that binds sFRP1. (C and D) IP analysis of Myc-sFRP1 (C) and HA-HSP90 (D) binding. (E) BMDMs were treated with sFRP1 plus HSP90-siRNA or control. HSP90 and sFRP1 interactions are confirmed in BMDM lysates by IP with anti-HSP90, followed by western blot analysis with anti-HSP90 and anti-sFRP1 antibody, respectively. (F) Representative IHC images of human nerves stained for HSP90 at 12 h after injury. (G) t-SNE plots of injured nerves marked by genes of HSP90 isoforms. Color key from orange to yellow indicated relative gene expression levels from high to low. (H–J) Representative IF staining and related quantification of TNF-α (red), F4/80 (red), and p65 (green) staining on BMDMs treated with sFRP1 plus HSP90-siRNA and controls. Biological replicates n = 3 with two technical replicates each. (K–M) BMDM phenotypic switch as revealed by flow cytometric quantification. FITC reflects F4/80-positive cells. PE reflects CD206-positive cells. APC reflects CD86-positive cells. N = 6 biological replicates. (N–P) TUBB3 staining on sciatic DRG neurons cocultured with macrophages for 4 days and 7 days and related quantification of axonal length. Scale bar, 100 μm. The start and the end of an axon were marked by red arrows. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons tests; ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ns, no significance. Data were presented as mean ± SD.

Article Snippet: Recombinant sFRP1 protein , MedChemExpress , Cat#HY-P73413.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Incubation, Labeling, Protein-Protein interactions, Control, Western Blot, Staining, Gene Expression

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Depletion of HSP90 in macrophages attenuated neuroinflammation and nerve degenerative changes exerted by sFRP1 (A) Hsp90aa flox/+ mice were bred with Lyz2-cre mice to generate macrophage-specific HSP90-deficient ( Hsp90aa flox/+ Lyz2-cre ) and littermate control ( Hsp90aa flox/+ ) mice. (B and C) Representative IF images of SCG10 staining and related quantification of sciatic nerves at 2 weeks post injury. The dashed line indicates the transection site. Scale bar, 500 μm. N = 6 mice. (D and E) Representative IF images of F4/80 staining (red) of sciatic nerves and related quantification of macrophages at 2 weeks post injury. Scale bar, 100 μm. N = 6 mice. (F–I) Double staining of TNF-α (red) and IL-1β (green) on nerve longitudinal sections and related quantification. (J–L) Representative TUBB3 staining (green) and related quantification of sciatic DRG neurons isolated from Hsp90aa flox/+ and Hsp90aa flox/+ Lyz2-cre mice after 4 days and 7 days of culture. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using two-way ANOVA followed by Sidak’s post hoc analysis in (C) and (E), and using two-tailed unpaired Student’s t tests in (F), (G), (K), and (L); ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗ p < 0.05; ns, no significance. Data were presented as mean ± SD.

Article Snippet: Recombinant sFRP1 protein , MedChemExpress , Cat#HY-P73413.

Techniques: Control, Staining, Double Staining, Isolation, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: SFRP1-neutralizing antibody treatment improved axon regeneration in vivo and in vitro (A and B) Representative SCG10 immunostaining and related quantification of murine injured nerves at 2 weeks after nerve transection. The dashed line indicates the transection site. Scale bar, 500 μm. N = 6 mice. (C) Schematic diagram of DRG neuron and macrophage microfluidic coculture chamber assay. (D) Representative optical images of macrophages in the neuron-macrophage coculture chambers. (E and F) Representative TUBB3 immunofluorescent images of neurons in the neuron-macrophage co-culture chambers and related quantification of average axon length in microfluidic channels. Biological replicates n = 3 with two technical replicates each. (G) Schematic diagram of DRG neuron and macrophage direct coculture assay. (H and I) Representative IF images stained for TUBB3 (green) on sciatic DRG neurons, and quantification of average axon length per cell in the direct coculture dishes. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using two-way ANOVA followed by Sidak’s post hoc analysis in (B) and (I) and using two-tailed unpaired Student’s t tests in (F); ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05. Data were presented as mean ± SD.

Article Snippet: Recombinant sFRP1 protein , MedChemExpress , Cat#HY-P73413.

Techniques: In Vivo, In Vitro, Immunostaining, Boyden Chamber Assay, Co-Culture Assay, Co-culture Assay, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet:

Article Snippet: Recombinant sFRP1 protein , MedChemExpress , Cat#HY-P73413.

Techniques: Cytometry, Recombinant, Sequencing, Plasmid Preparation, Software

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: LepR+ stromal cells respond to periodontitis and attenuate alveolar bone repair via CCRL2 mediated Wnt inhibition.

doi: 10.1093/jbmr/zjae036

Figure Lengend Snippet: Figure 4. The Wnt/β-catenin signaling in LepR+/CAR cells downregulated via CCRL2 in the presence of inflammation. (A) PCA of the RNA-Seq data of LepR+/CAR cells derived from WT and Ccrl2-KO mice after osteogenic induction and TNFα stimulation for 2 d. (B) Volcano plot showing differential gene expression in LepR+/CAR cells of WT and Ccrl2-KO mice. (C) Heatmap of top25 upregulated genes in LepR+/CAR cells of Ccrl2-KO mice compared with WT mice. (D) Heatmap of top25 downregulated genes in LepR+/CAR cells of Ccrl2-KO mice compared with WT mice. (E, F) GO-BP and KEGG enrichment analysis of the upregulated differentially expressed genes of LepR+/CAR cells of Ccrl2-KO mice compared with WT mice. (G, H) Western blot analysis (n = 3) of CCRL2, SFRP1, Wnt/β-catenin signaling-related proteins, and OSX expression levels with or without TNFα stimulation in LepR+/CAR cells of WT and Ccrl2-KO mice. (I) Immunofluorescence staining of LepR and SFRP1 in the extraction sockets of Ccrl2-KO and WT mice 3 d postextraction (scale bar: left, 120 μm; right, 15 μm). (J) Immunofluorescence staining of LepR and active β-catenin in the extraction sockets of Ccrl2-KO and WT mice 3 d postextraction (scale bar: left, 120 μm; right, 15 μm). (K) Immunofluorescence staining of SFRP1 in human healthy gingival tissues, periodontitis gingival tissues, and periodontitis extraction socket tissues (scale bar: left, 80 μm; right, 15 μm). (L) Percentage of SFRP1+LepR+/LepR+ cells in the extraction sockets of Ccrl2-KO and WT mice (n = 6). (M) Percentage of active β-catenin+LepR+/LepR+ cells in the extraction sockets of Ccrl2-KO and WT mice (n = 6). (N) Percentage of SFRP1+/DAPI+ cells in healthy gingival tissues, periodontitis gingival tissues, and periodontitis extraction socket tissues (n = 6).

Article Snippet: In the recombinant SFRP1 (rSFRP1)-treated group, 1 μg/mL recombinant mouse SFRP1 protein (R&D Systems, USA) was added to the osteogenic induction medium.

Techniques: RNA Sequencing, Derivative Assay, Gene Expression, Western Blot, Expressing, Staining, Extraction

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: LepR+ stromal cells respond to periodontitis and attenuate alveolar bone repair via CCRL2 mediated Wnt inhibition.

doi: 10.1093/jbmr/zjae036

Figure Lengend Snippet: Figure 5. The CCRL2 in LepR+/CAR cells inhibits osteogenesis by binding to SFRP1 to suppress Wnt/β-catenin signaling under inflammation. (A) Coimmunoprecipitation analysis of the interaction between CCRL2 and SFRP1 in LepR+/CAR cells of Ccrl2-KO and WT mice with or without TNFα stimulation. (B) GST pull-down assay confirmed the binding of recombinant GST-SFRP1 and CCRL2 derived from the lysates of LepR+/CAR cells under TNFα induced inflammation. (C) CCRL2 and SFRP1were coimmunoprecipitated using Wnt3a antibody in LepR+/CAR cells of Ccrl2-KO and WT mice with or without TNFα stimulation. (D, E) Western blot analysis (n = 3) of the changes in CCRL2, Wnt/β-catenin signaling-related proteins, and OSX expression levels in LepR+/CAR cells of Ccrl2-KO and WT mice with or without TNFα stimulation in the presence of rSFRP1. (F) Statistical analysis (n = 3) of the inhibition rates of active β-catenin, p-GSK3β-Ser9, and OSX protein expression in LepR+/CAR cells of Ccrl2-KO and WT mice with TNFα stimulation in the presence of rSFRP1. (G, H) ALP staining and analysis (n = 3) of ALP activity after osteogenic induction for 7 d in LepR+/CAR cells of Ccrl2-KO and WT mice with TNFα stimulation in the presence of rSFRP1. (I, J) ARS staining and OD value test (n = 3) after 21 d of osteogenic induction in LepR+/CAR cells of Ccrl2-KO and WT mice with TNFα stimulation in the presence of rSFRP1. (K, L) Western blot analysis (n = 3) of the changes in Wnt/β-catenin signaling-related proteins and OSX expression levels in LepR+/CAR cells of Ccrl2-KO and WT mice with or without Wnt3a stimulation in the presence of TNFα. (M) Statistical analysis (n = 3) of the activation rates of active β-catenin, p-GSK3β-Ser9, and OSX protein expression in LepR+/CAR cells of Ccrl2-KO and WT mice with TNFα stimulation in the presence of Wnt3a.

Article Snippet: In the recombinant SFRP1 (rSFRP1)-treated group, 1 μg/mL recombinant mouse SFRP1 protein (R&D Systems, USA) was added to the osteogenic induction medium.

Techniques: Binding Assay, Pull Down Assay, Recombinant, Derivative Assay, Western Blot, Expressing, Inhibition, Staining, Activity Assay, Activation Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: LepR+ stromal cells respond to periodontitis and attenuate alveolar bone repair via CCRL2 mediated Wnt inhibition.

doi: 10.1093/jbmr/zjae036

Figure Lengend Snippet: Figure 6. Inhibiting SFRP1 signaling in LepR+/CAR cells promotes alveolar bone healing in extraction sockets with periodontitis. (A) Schematic diagram of the experimental procedure to inject the SFRP1 inhibitor WAY-316606 into the extraction sockets of LepR-Cre; tdTomato mice. (B) Micro-CT images of extraction sockets in the vehicle and WAY-316606 group 14 d postextraction; sagittal view (upper) and coronal view (lower). Dashed lines indicate extraction sockets and the arrows indicate extraction sockets with periodontitis (scale bar: 200 μm). (C) Micro-CT analysis (n = 6) of bone healing in the vehicle and WAY-316606 groups. (D) Immunofluorescence staining of CCRL2 and OSX in the extraction sockets of the vehicle and WAY-316606 groups 3 d postextraction. LepR-td represents LepR+/CAR cells (scale bar: left, 120 μm; right, 15 μm). (E) Immunofluorescence staining of active β-catenin in the extraction sockets of the vehicle and WAY-316606 groups 3 d postextraction. LepR-td represents LepR+/CAR cells (scale bar: left, 120 μm; right, 15 μm). (F) Percentage of OSX+ LepR-td+/LepR-td+ cells in the vehicle and WAY-316606 groups. (G) Percentage of CCRL2+ LepR-td+/LepR-td+ cells in the vehicle and WAY-316606 groups. (H) Percentage of active β-catenin+LepR-td+/LepR-td+ cells in the vehicle and WAY-316606 groups. (I) Schematic diagram illustrating the mechanism by which CCRL2 inhibits the osteogenic differentiation of LepR+/CAR cells during tooth extraction socket healing with periodontitis. (n = 6 for each group.)

Article Snippet: In the recombinant SFRP1 (rSFRP1)-treated group, 1 μg/mL recombinant mouse SFRP1 protein (R&D Systems, USA) was added to the osteogenic induction medium.

Techniques: Extraction, Micro-CT, Staining

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Wnt1 Boosts Fracture Healing by Enhancing Bone Formation in the Fracture Callus.

doi: 10.1002/jbmr.4797

Figure Lengend Snippet: Fig. 7. Effects of recombinant Wnt1 on critical-size bone defect healing. (A) Experimental design. (B) Representative 3D reconstructions from μCT datasets. The entire area between the two inner pin holes in shown. Red indicates less mineralized bone, whereas yellow and blue indicate higher mineralized bone. (C) Bone volume in the defect area. (D) Polar moment of inertia of the bone in the defect area as analyzed by μCT evaluation. (E) Trabecular thickness, (F) trabecular separation, and (G) trabecular number of the newly formed bone in the defect area. (H) Active YAP1 and BMP2 immunostaining in the defect area. Scale bar = 100 μm. n = 7 per group. Mann-Whitney U test. sFRP1, secreted frizzled-related protein 1.

Article Snippet: The critical-size model has been described. (31) Five micrograms (5 μg) of recombinant sFRP1 protein alone (R&D Systems; 9019-SF-025) was used for the control group.

Techniques: Recombinant, Immunostaining, MANN-WHITNEY