sfrp1 2  (MedChemExpress)

Journal: Nature Communications

Article Title: The pericardium forms as a distinct structure during heart formation

doi: 10.1038/s41467-025-63599-5

Figure Lengend Snippet: A – E Ventral confocal imaging (max projection) of representative 72 hpf hand2:EGFP;myl7DsRed larvae undergoing distinct treatments as indicated; anterior to the top; v ventricle, a atrium. A , B Representative larvae treated with DMSO vehicle only as control at 18 hpf overnight showing hand2:EGFP- expressing pericardial sac surrounding the heart at 72 hpf ( A , 20x) and cellular density ( B , 40x zoom, representative nuclei marked with dashed lines). C , D Ventral images of representative hand2:EGFP;myl7:DsRed larvae treated with the Wnt signaling inhibitor IWR-1 at 18 hpf overnight, showing expanded pericardial sac and edema with large, stretched cells surrounding the larval zebrafish heart at 72 hpf ( C 20x) and lower cellular density ( D 40x). E , F Ventral images of hand2:EGFP;myl7:DsRed larvae treated with BDM as myosin II inhibitor at 18 hpf overnight showing expanded pericardial sac with normal cell distribution at 72 hpf ( E 20x) and cellular density ( F 40x). G – J Quantifications of pericardial and cardiac features following the treatments. One-way ANOVA, n = 6 animals, three independent experiments. G Heart rate of vehicle-treated, Wnt-inhibited, and myosin II-inhibited (BDM) animals ( p = 0.6056 DMSO to IWR-1, p = 0.0001 DMSO to BDM). H Pericardial area (distribution per ventral view), showing increased pericardial area in IWR-1-treated animals ( p = 0.0631 DMSO to IWR-1, p = 0.317- DMSO to BDM). I Cell density (cells per square millimeter), showing decreased cell density in IWR-1-treated animals only ( p = 0.0001 DMSO to IWR-1, p = 0.01345 DMSO to BDM). J Cell size showing increases in IWR-1-treated embryos only ( p = 0.0001 DMSO to IWR-1, p = 0.9918 DMSO to BDM). K , L Increased tissue stiffness in the pericardium of rats treated with PBS (vehicle), Iso only, sFRP1 only, or combined Isoproterenol (Iso) and SFRP1 ( n = 3 per condition). Neonatal rats (0-to-4-day old rats) were injected intraperitoneally with 0.05 mg/kg/day of human recombinant sFRP1 protein and Iso in an animal model of pediatric dilated cardiomyopathy. Atomic force microscopy (AFM) of dissected pericardia provided measures for Young’s modulus (kPa) as readout for tissue elasticity, with treated pericardia showing increased stiffness with combined Iso and sFRP1 only ( K ) as quantified per sample( L , unpaired two-tailed t -test, p = 0.9093 vehicle to Iso only, p = 0.6129 vehicle to sFRP1 only, p = 0.0140 vehicle to Iso + sFRP1). Each dot represents an individual sample. Representative images of control and = treated rats. Source data are provided as a Source Data file. Scale bar A , C , E 200 μm; B , D , F (40x) 50 μm. Species silhouettes were adapted from the PhyloPic database ( https://www.phylopic.org/ ).

Article Snippet: The sFRP1 (Recombinant Human sFRP1 Protein, CF, R&D systems) solution was freshly prepared for each treatment by dissolving in phosphate-buffered saline (PBS) at room temperature.

Techniques: Imaging, Control, Expressing, Injection, Recombinant, Animal Model, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Cross-species comparison reveals therapeutic vulnerabilities halting glioblastoma progression

doi: 10.1038/s41467-025-62528-w

Figure Lengend Snippet: a Shared ptalign axis between GBMs and v-SVZ enables comparative assessment of healthy vs malignant expression dynamics by pairwise EMD. Mean EMD across GBMs informs gene dysregulation among the GBM cohort. b Genes ranked by mean EMD vs v-SVZ among n = 51 GBMs. Genes above the inflection point ( n = 164, purple) are considered recurrently dysregulated. c GO enrichment of recurrently dysregulated genes from ( b ) with all considered genes as background. P -values by hypergeometric test with FDR-correction. d Zoomed view of recurrently dysregulated genes from ( b ) with key signaling pathway genes indicated. e v-SVZ expression bias for recurrently dysregulated genes from ( b ), colored by pathway genes from ( d ). P -values represent a one-sided permutation test for Q-bias. f Expression splines and EMD values for individual genes from ( d ). Individual, and 95% confidence intervals, of mean GBM expression dynamics are colored red, while v-SVZ dynamics are blue. g Log-normalized SFRP1 expression in the v-SVZ UMAP. Cycling cells are colored gray. Source data are provided as a Source Data file.

Article Snippet: Human SFRP1 (Origene plasmid #RC207328) or Notum (Gateway ORF clone ID #164485821) was cloned in frame under 7xTCF promoter (7TGC; Addgene plasmid #24304) upstream of EGFP sequence using In-Fusion HD cloning kit (Takara) according to the manufacturer’s instructions (Supplementary Data ).

Techniques: Expressing

Journal: Nature Communications

Article Title: Cross-species comparison reveals therapeutic vulnerabilities halting glioblastoma progression

doi: 10.1038/s41467-025-62528-w

Figure Lengend Snippet: a SFRP1-OE lentiviral construct used to generate GBM PDXs. mCherry ubiquitously labels tumor cells. Created in BioRender. Kaya, O. (2025) https://BioRender.com/np19rtk . b Kaplan–Meier curve of mice reaching endpoint post injection among three batches of n = 6 for control and SFRP1-OE mice each. P -value from log-rank test, precisely 1.3 × 10 −4 . c Proportion of QAD-stage cells identified by ptalign in SFRP1-OE ( n = 3 replicates) and control ( n = 4 replicates) scRNA-seq. Bars present mean, error bars standard deviation. d Selected GSEA enrichments from genes ranked by DEseq2 log fold-change between pseudobulked SFRP1-OE and control from ( c ). P -values from GSEA enrichment are FDR-adjusted. e Parameter estimates from population models of GBM dynamics (Fig. ), including activation- and inferred growth rates, as well as self-renewal and amplification probabilities for T6 control and SFRP1-OE GBM PDXs from ( c ) among n = 51 primary GBMs. The rank percentile for each parameter is indicated. f Representative immunofluorescence images of GBM cells in a control and SFRP1-OE ( e ) PDX brain. Scale bars 100 µm, in insets 25 µm. g Entire spatial transcriptomics ROI depicting similar regions in SFRP1-OE ( g ) and control PDX brains. Transcripts were associated with segmented nuclei to assign species and QAD-stage. Pie charts indicate the sum of QAD-stage cells by brain region across ROIs. ROI region of interest, CTX cortex, CC corpus callosum, LV lateral ventricle, V-outgr. ventricular outgrowth, SN septal nuclei, STR striatum. Source data are provided as a Source Data file.

Article Snippet: Human SFRP1 (Origene plasmid #RC207328) or Notum (Gateway ORF clone ID #164485821) was cloned in frame under 7xTCF promoter (7TGC; Addgene plasmid #24304) upstream of EGFP sequence using In-Fusion HD cloning kit (Takara) according to the manufacturer’s instructions (Supplementary Data ).

Techniques: Construct, Injection, Control, Standard Deviation, Activation Assay, Amplification, Immunofluorescence

Journal: Nature Communications

Article Title: Cross-species comparison reveals therapeutic vulnerabilities halting glioblastoma progression

doi: 10.1038/s41467-025-62528-w

Figure Lengend Snippet: a Cross-species interrogation of murine v-SVZ cell type VMRs from Kremer et al. . Example NSC and astrocyte VMR methylation profiles are depicted, with methylation at the corresponding human locus quantified in SFRP1-overexpressing (OE) and control GBM WGBS. b Left: Mean methylation of v-SVZ cell type-specific VMRs from ref. in SFRP1-OE and control WGBS ( n = 3 technical replicates each). Differentially methylated regions are highlighted by genotype. Right: v-SVZ cell type VMRs in a Gaussian KDE over the vertical axis of the scatterplot. P -value by one-sided hypergeometric test, for Astro. precisely 5.8 × 10 −5 . c Selected v-SVZ astrocyte VMR overlapping an NFIB-promoter in SFRP1-OE and control. Points depict mean CpG methylation among replicates, lines comprise a 10-CpG moving average. d Proportion of NFIB+ (left) and GFAP+ (right) Q-cells among control and SFRP1-OE samples. P -value by two-sided t -test, precisely 1.4 × 10 −4 and 3.4 × 10 −5 for NFIB, GFAP, respectively. e Schematic representation of the lineage potential in NSC-like GBM cells in control (above) vs lineage restrictions imposed by the remodeled methylome of the expanded astrocyte-like GBM cells upon SFRP1-OE (bottom). Adapted from ref. , Springer Nature, Inc. VMR: variably methylated region. Source data are provided as a Source Data file.

Article Snippet: Human SFRP1 (Origene plasmid #RC207328) or Notum (Gateway ORF clone ID #164485821) was cloned in frame under 7xTCF promoter (7TGC; Addgene plasmid #24304) upstream of EGFP sequence using In-Fusion HD cloning kit (Takara) according to the manufacturer’s instructions (Supplementary Data ).

Techniques: Methylation, Control, CpG Methylation Assay

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: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5μL) or PBS (5μL), immediately after nerve transection procedure.

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: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5μL) or PBS (5μL), immediately after nerve transection procedure.

Techniques: Gene Expression, 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: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5μL) or PBS (5μL), immediately after nerve transection procedure.

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: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5μL) or PBS (5μL), immediately after nerve transection procedure.

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: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5μL) or PBS (5μL), immediately after nerve transection procedure.

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: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5μL) or PBS (5μL), immediately after nerve transection procedure.

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: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5μL) or PBS (5μL), immediately after nerve transection procedure.

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: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5μL) or PBS (5μL), immediately after nerve transection procedure.

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

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: For sFRP1 inhibition treatments, WAY-316606 (HY-10858, MedChemExpress, 500nM, 5μL) and LPS (15 mg/kg) were administered into different mice through intraneural injection, while control mice received PBS injection.

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: For sFRP1 inhibition treatments, WAY-316606 (HY-10858, MedChemExpress, 500nM, 5μL) and LPS (15 mg/kg) were administered into different mice through intraneural injection, while control mice received PBS injection.

Techniques: Gene Expression, 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: For sFRP1 inhibition treatments, WAY-316606 (HY-10858, MedChemExpress, 500nM, 5μL) and LPS (15 mg/kg) were administered into different mice through intraneural injection, while control mice received PBS injection.

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: For sFRP1 inhibition treatments, WAY-316606 (HY-10858, MedChemExpress, 500nM, 5μL) and LPS (15 mg/kg) were administered into different mice through intraneural injection, while control mice received PBS injection.

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: For sFRP1 inhibition treatments, WAY-316606 (HY-10858, MedChemExpress, 500nM, 5μL) and LPS (15 mg/kg) were administered into different mice through intraneural injection, while control mice received PBS injection.

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: For sFRP1 inhibition treatments, WAY-316606 (HY-10858, MedChemExpress, 500nM, 5μL) and LPS (15 mg/kg) were administered into different mice through intraneural injection, while control mice received PBS injection.

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: For sFRP1 inhibition treatments, WAY-316606 (HY-10858, MedChemExpress, 500nM, 5μL) and LPS (15 mg/kg) were administered into different mice through intraneural injection, while control mice received PBS injection.

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: For sFRP1 inhibition treatments, WAY-316606 (HY-10858, MedChemExpress, 500nM, 5μL) and LPS (15 mg/kg) were administered into different mice through intraneural injection, while control mice received PBS injection.

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