recombinant mouse spp1 protein  (R&D Systems)

Journal: Cell Regeneration

Article Title: Integrative analysis and experimental validation identify the role of CD44 and Nucleolin in regulating gliogenesis following spinal cord injury

doi: 10.1186/s13619-025-00253-x

Figure Lengend Snippet: Dynamic cell portion changes and intercellular communication analysis revealed SPP1 signaling pathway was critical in microglia after SCI. A , B Stacked bar plots depicting changes in the relative abundance of major cell types in spinal cord ( A ) and peripheral immune cell populations ( B ) across various time points. Astrocytes, microglia, OPCs, and MDMs show marked shifts, particularly in the acute (1~3 dpi) and subacute phases of SCI, the absence of 42 dpi stems directly from the source data ( GSE172167 ), where immune cell clusters were not identified or annotated at this specific time point in the original study. C Intercellular communication networks illustrate increased signaling complexity at 1 and 3 dpi compared to the sham condition. D Quantitative result of the total number of interactions in sham, 1 dpi, and 3 dpi samples, showing a significant increase in cell–cell interactions post-injury. E Heatmaps displaying the changes in signaling patterns for key cell types. SPP1 became prominent at 1 dpi. F Information flow of microglia indicated the SPP1 signal was significant

Article Snippet: After adhesion, recombinant mouse SPP1 protein (MCE, Cat No. HY- P71786 ) and recombinant mouse PTN protein (MCE, Cat No. HY- P71213 ) were separately administered to the microglia and astrocytes at concentrations of 0, 0.1, 0.5, and 1 μg/mL for a duration of 24 h. Subsequent to the stimulation period, the culture medium was carefully removed, and the cells were gently washed twice with PBS.

Techniques:

Journal: Cell Regeneration

Article Title: Integrative analysis and experimental validation identify the role of CD44 and Nucleolin in regulating gliogenesis following spinal cord injury

doi: 10.1186/s13619-025-00253-x

Figure Lengend Snippet: SPP1-CD44 signaling promotes microglial activation and inflammatory response. A SPP1 signaling pathway network showing interactions between microglia and other cell types. B A circular plot illustrating the interaction network of microglia with other cells in various ligand -receptor pairs, including Spp1 - Cd44 . C , D Violin plots showing expression levels of Spp1 and Cd44 across different cell types in sham (blue) and 1 dpi (red). Both genes show elevated expression in microglia following injury. E Violin plot of Cd44 expression in microglia subclusters, showing the upregulation in the wound healing and inflammatory response2 cluster at 1 dpi. F The microglia were sorted by flow cytometry and ( G ) Cd44 gene expression was detected by qRCR. H Flow cytometry analysis of CD44 positive microglia after SCI, showing a marked increase in CD44 + microglia during 7 dpi. I Immunofluorescence images of spinal cord lesion site stained for Iba1, CD44, SPP1, and merged with DAPI. White arrows indicate the co-stained CD44 + and SPP1 + signals in Iba1 positive microglia ( J ) Quantification results of CD44 + and SPP1 + in Iba1 positive microglia cells. K Using PLA to detect specific SPP1-CD44 interactions of spinal cord lesion site in situ. L Quantification of PLA results, the PLA signal is quantified and plotted as the area of PLA signal per Iba1 positive cell. M , N qRT-PCR showing dose- and time-dependent increases of Cd44 expression in BV2 microglia after recombinant SPP1 stimulation. O Representative images of PLA assay specific SPP1-CD44 interactions of BV2 microglia cells in vitro. P PLA signal was quantified and plotted as the area of PLA signal per cell. Q ELISA quantification of IL-6 levels in cell supernatant after SPP1 stimulation. R Western blot showing the time course of CD44 and p-NF-κB p65 protein expression in BV2 cells after SPP1 treatment. ( S – T ) Quantification of CD44 and p-NF-κB p65 protein levels. Data are presented as mean ± SEM. ( n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: After adhesion, recombinant mouse SPP1 protein (MCE, Cat No. HY- P71786 ) and recombinant mouse PTN protein (MCE, Cat No. HY- P71213 ) were separately administered to the microglia and astrocytes at concentrations of 0, 0.1, 0.5, and 1 μg/mL for a duration of 24 h. Subsequent to the stimulation period, the culture medium was carefully removed, and the cells were gently washed twice with PBS.

Techniques: Activation Assay, Expressing, Flow Cytometry, Gene Expression, Immunofluorescence, Staining, In Situ, Quantitative RT-PCR, Recombinant, In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) Scheme of human cancer types included in the integrated analysis. (B) Numbers of samples in each normal tissue or cancer type. (C) Marker genes of the major cell types in the pan-cancer scRNA-seq atlas. (D) UMAPs showing LUM , MCAM , CSPG4 , MYH11 , ACTA2 , FAP , MMP11 , PIEZO2 , C1QA , C1QB , C1QC , CD34 expression in CAFs. (E) Signature genes of apCAF subclusters. (F) Expression of CD24 and CD37 in the two apCAF lineages. (G) Combined overall survival of the 14 types of cancer with SPP1 expression. (H) Regulon of SPI1 in each CAF subcluster revealed by SCENIC algorithm. (I) Regulon of POU5F1 in each CAF subcluster revealed by SCENIC algorithm. (J) ChIP-seq binding peaks of OCT4 at the SPP1 promoter in fibroblasts visualized using the WashU Epigenome Browser from the Cistrome project.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Marker, Expressing, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) All apCAFs marked by MHC II molecule expression are extracted from and re-clustered, revealing four apCAF subclusters. (B) UMAPs of signature genes of apCAF subclusters including CD74 , MSLN , UPK3B , KRT19 , PTPRC , CD52 (C) Pseudotime analysis reveals two distinct trajectories of apCAFs. Expression of CD74 , HLA-DRA , MSLN , PTPRC and SPP1 along the trajectories are shown. (D) Up-regulated genes in the F-apCAF lineage (subcluster 2 vs 1) are used to perform GSEA pathway analysis. Significant pathways are shown. (E) Up-regulated genes in the M-apCAF lineage (subcluster 3 vs 0) are used to perform GSEA pathway analysis. Significant pathways are shown. (F) Differentially expressed genes in apCAFs in cancer compared to normal tissues. Six most up-regulated and robustly expressed genes are identified: NDUFA4L2 , SPP1 , PLOD2 , EGLN3 , ANGPTL4 , HILPDA . (G) Expression of NDUFA4L2 , SPP1 , PLOD2 , EGLN3 , ANGPTL4 , HILPDA in each CAF subcluster. (H) Combined overall survival of the 14 types of cancer with the six-gene signature ( NDUFA4L2 , SPP1 , PLOD2 , EGLN3 , ANGPTL4 , HILPDA ). (I) Regulatory network of transcription factors in each CAF subcluster revealed by SCENIC algorithm. (J) Regulatory network of genes by SPI1 in F-apCAFs. (K) Regulatory network of genes by POU5F1 in M-apCAFs. (L) Abundance of F-apCAFs in different cancer types. (M) Abundance of M-apCAFs in different cancer types.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Expressing

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) IHC staining for pan-cytokeratin (PanCK) in human PM samples. Scale bars, 250 μm. (red arrow, normal mesothelium; blue arrow, cytokeratin + CAFs). (B) Multiplex IHC staining for PanCK, SPINK4 and DAPI in human PM samples. Scale bars, 10 μm. (C) Visualization of normal mesothelium adjacent to M-apCAF-enriched areas from Xenium assay. M-apCAFs, cancer cells and the expression of normal mesothelial cell genes MSLN and UPK3B are shown. (D) Ligand-receptor interaction analysis between M-apCAFs (ligands) and different populations of immune cells (receptors). (E) Ligand-receptor interaction analysis between M-apCAFs (ligands) and cancer cells (receptors). (F) SPP1 expression in the iCMS2 CAFs (patient 2, 3, 5, 8 (P2, P3, P5, P8)) and iCMS3 CAFs (patient 1, 4, 6, 7 (P1, P4, P6, P7)) from the GeoMx assay.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Immunohistochemistry, Multiplex Assay, Expressing

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) Robust cell type decomposition is performed in human PM sample with robust cytokeratin + CAF formation to deconvolve the Xenium data into cell types using our pan-cancer scRNA-seq atlas as reference. (B) Four spatial niches are identified. Percentages of cell types within each niche are shown. (C) Visualization of the spatial distribution of different cell types in four M-apCAF-enriched regions. (D) Expression of T cell immunosuppressive genes across four spatial niches. (E) RT-PCR (n=3/group) and western blots measuring the expression of SPP1 in OmMeso cells after tumor conditioned medium treatment. (F) Wound healing assays are performed to measure the migration capability of MC38 colon cancer cells in the presence of mouse recombinant protein or anti-SPP1 mAb. Representative pictures of cell migration at 0h, 24h, 48h are shown. n=3/group. (G) Matrigel transwell assays in the presence of mouse recombinant protein or anti-SPP1 mAb for 24 hours are performed. Representative pictures for each group are shown. n=3/group. (H) MC38 cancer cells are injected intraperitoneally into wildtype (WT) or Spp1 knockout (KO) mice on a C57BL/6 background (WT, n=8; KO, n=10). Mice are sacrificed 4 weeks after cancer cell injection. Peritoneal cancer index (PCI) scores and ascites formation are measured. (I) MC38 cancer cells are injected intraperitoneally into wildtype C57BL/6 mice. Mice are treated with control Ab or anti-SPP1 mAb (n=5/group) one week after cancer cell injection and maintained at two doses/week. Mice are sacrificed 4 weeks after cancer cell injection. PCI scores and ascites formation are measured.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Migration, Recombinant, Injection, Knock-Out, Control

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) UMAP of major cell types across the 6 human PDAC samples identified from the Xenium assays. (B) Marker genes of the major cell types in PDAC. (C) Proportions of the major cell types in the treatment naïve and chemoradiotherapy (chemo-RT)-treated PDAC samples. (D) Expression of SPP1 in cancer cell 1 and cancer cell 2 populations. (E) Robust cell type decomposition is performed in PDAC to deconvolve the Xenium data into cell types using our pan-cancer scRNA-seq atlas as reference. (F) Spp1 WT or KO tumors are digested into single cell suspension and subjected to scRNA-seq. Major cell types are identified in the merged data from Spp1 WT and KO groups. (G) Marker genes of major cell types in Spp1 WT and KO tumors. (H) IHC staining and quantification for CD3 and CD8 in Spp1 WT and KO tumors (n=3/group). (I) Expression of Spp1 , Ptprc , Cd24a and Msln in CAFs of Spp1 WT and KO tumors.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Marker, Expressing, Suspension, Immunohistochemistry

Journal: bioRxiv

Article Title: Single-cell resolution spatial analysis of antigen-presenting cancer-associated fibroblast niches

doi: 10.1101/2024.11.15.623232

Figure Lengend Snippet: (A) Spatial niches are identified in human PDAC sample with TLS formation. (B) Based on the spatial niches and expression of SPP1 in cancer cells, PDAC sample is classified into four areas: stroma, TLS, SPP1 - and SPP1 + cancer. Deconvolved cell types are shown in each area. (C) Expression of SPP1 is visualized in SPP1 - and SPP1 + cancer areas. (D) Proportions of F-apCAFs and M-apCAFs are quantified in stromal, TLS, SPP1 - and SPP1 + cancer areas (n=3 for each area). (E) Western blots measuring the expression of SPP1 in PanMeso cells after tumor conditioned medium treatment. (F) GFP + PanMeso cells are co-injected with a murine PDAC cell line (BMFA3: In Vivo 1 or CT1BA5: In Vivo 2) at a 1:1 ratio. Tumors are harvested 1 month after injection and digested into single-cell suspension. GFP + cells are collected by flow sorting and subjected to RNA-seq analysis in comparison to parental PanMeso cells to evaluate the Spp1 expression. (G) Syngeneic PDAC cancer cells (6620c1) are injected orthotopically into wildtype (WT) or Spp1 knockout (KO) C57BL/6 mice (n=6/group). Tumors are harvested 1 month after injection. (H) Spp1 WT or KO tumors are digested into single cell suspension and subjected to scRNA-seq (6 tumors/group, every two tumors are pooled together for library construction). Ratio of each cell type between WT and KO group is compared and quantified. (I) CAFs from both Spp1 WT or KO tumors are extracted from the scRNA-seq data. iCAF, myCAF and apCAF clusters are identified. (J) Signature genes of each CAF subtype. (K) Proportional changes of CAF subtypes between Spp1 WT and KO group. (L) UMAPs showing sslCAF marker Pi16 and Dpt expression between Spp1 WT and KO tumors. (M) CytoTRACE analysis determining the progenitor and differentiation status among iCAFs, myCAFs and apCAFs, with higher score indicating more stem-like and less differentiated status. (N) Quantification of the expression of T cell chemoattractant genes in CAFs between Spp1 WT and KO tumors. (O) Syngeneic PDAC cancer cells (6620c1) are injected orthotopically into wildtype C57BL/6 mice. Mice are treated with control Ab (n=5) or anti-SPP1 mAb (n=7) one week after cancer cell injection and maintained at two doses/week. Mice are sacrificed 4 weeks after cancer cell injection.

Article Snippet: For the wound healing assay, cells were plated onto 6-well tissue culture plates coated with 50 μg/ml Matrigel (BD Biosciences) with or without 100 ng/ml recombinant mouse SPP1 protein (R&D Systems) or 1 μg/ml SPP1 monoclonal antibody (Bio X Cell).

Techniques: Expressing, Western Blot, Injection, In Vivo, Suspension, RNA Sequencing Assay, Comparison, Knock-Out, Marker, Control

Journal: Aging Cell

Article Title: Single‐cell RNA sequencing reveals the CRTAC1 + population actively contributes to the pathogenesis of spinal ligament degeneration by SPP1 + macrophage

doi: 10.1111/acel.14320

Figure Lengend Snippet: Characterization of ligament immune compartment and possible pathogenic role of SPP1 + macrophages. (a) UMAP plot of immune cells in spinal ligament tissues. (b) Inflammation scores of the main immune cell lineages in ligament. (c) UMAP plot of monocytes and macrophages in spinal ligament tissues. (d) Hierarchical clustering of DEGs in monocytes and macrophages. (e) Pseudo‐time analysis of monocyte and macrophage subpopulations. (f) Pathway enrichment analysis for DEGs of macrophages from ligament tissues. (g) Ligand–receptor interactions between macrophages and other cell types. (h–j) Quantitative PCR assays (h) and western blot analysis (i, j) of control cultured primary ligament cells and those treated with short interfering RNA targeting ATF3 and recombinant SPP1. *, p ‐value < 0.05; **, p ‐value < 0.01; ***, p ‐value < 0.001.

Article Snippet: For recombinant protein stimulation experiments, primary human ligament cells from patients with traumatic injury were incubated with 200 ng/mL recombinant SPP1 protein (1433‐OP‐050, R&D Systems, USA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control, Cell Culture, Small Interfering RNA, Recombinant

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Schematic diagram illustrating the experimental procedure for diet-induced obesity (DIO) mice model. 9-week-old Esrra fl/fl and Esrra AKO male mice were fed a normal chow diet (NCD) or high-fat diet (HFD) for 16 weeks. (Schematic created with BioRender.com. Agreement number: WP26KB8FER). b Representative pictures and body weights of NCD and DIO mice. c Representative images and weight analysis of white adipose tissue (WAT) depots, including gonadal WAT (gWAT), inguinal WAT (iWAT), and mesentery WAT (mWAT). d Plasma triglyceride (TG) and free fatty acid (FFA) levels. e Plasma leptin, IL-6 and TNFa levels. f Representative micro-CT images of the distal femoral trabecular bone. g Quantitative analysis of bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N) and trabecular separation (Tb. Sp). h H&E staining of femur sections (scale bar: 100 μm). Yellow arrows indicate the bone marrow adipocytes. Osteoblast surface to bone surface ratio (Ob.S/BS) and osteoblast number to bone surface ratio (Ob.N/BS) are shown on the right panel. i Calcein double labeling of trabecular bone (scale bar: 50 μm). Mineral apposition rate (MAR) and bone formation rate (BFR/BS) were determined as graphs. j TRAP staining of femur sections with quantitative analysis of Oc.S/BS and Oc.N/BS. TRAP‐positive purple spots indicate multinucleated osteoclasts (scale bar: 100 μm). Plasma P1NP ( k ) and CTX1 ( l ) levels. m PLIN1 positive marrow adipocytes (PLIN1 + , red) and SPP1 (green) immunofluorescence staining in femur sections (scale bar: 100 μm). The box in the upper showing the metaphysis region near growth plate is represented at higher magnification in the bottom (scale bar: 50 μm). n The number and area of adipocytes in the femur marrow per tissue area and the quantification of SPP1 fluorescence intensity were measured from femur sections in ( m ). Data are shown as mean ± SD ( n = 6 mice per group). * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using two-way ANOVA with Fisher’s LSD post hoc analysis. Source data are provided as a Source Data file.

Article Snippet: Wild-type BMSCs were differentiated in the mixed induction medium supplemented with 10% WAT-CM or BMAds-CM as indicated, in the presence of 0.5 μg/ml recombinant SPP1 protein (rSPP1, Abcam #ab281820), 1 μg/ml SPP1 neutralizing antibody (SPP1 Nab, Novus Biologicals #AF808), 0.5 μg/ml recombinant leptin protein (rLeptin, R&D Systems #490-OB-01M), 250 nM leptin receptor antagonist Allo-aca (MCE #HY-P3212) or an equal volume of vehicle IgG (Cell Signaling Technology #2729).

Techniques: Micro-CT, Staining, Labeling, Immunofluorescence, Fluorescence

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Schematic diagram illustrating the experimental procedure for ovariectomy (OVX)-induced osteoporosis mice model. 10-week-old Esrra fl/fl and Esrra AKO female mice underwent either sham or OVX operation for 8 weeks (schematic created with BioRender.com. Agreement number: II26KB8K2T). b Representative images and weights of adipose depots. c Representative images and adipocytes size analysis from H&E-stained gWAT sections (scale bar: 50 μm). d Plasma leptin levels. Micro-CT images of distal femurs in sham and OVX mice ( e ) with morphometric analysis of BV/TV, Tb.N, Tb.Th, and Tb.Sp ( f ). g Representative TRAP-stained images and quantification of Oc.S/BS and Oc.N/BS in distal femoral metaphysis regions from sham and OVX mice (scale bar: 100 μm). h Representative H&E-stained images and quantification of Ob.S/BS and Ob.N/BS (scale bar: 100 μm). Plasma P1NP ( i ) and CTX1 ( j ) levels. k Calcein double labeling with quantitative analysis of MAR and BFR/BS (scale bar: 50 μm). l , m Immunofluorescence co-staining and quantification of PLIN1 + bone marrow adipocytes (red) and SPP1 (green) of femur sections from sham and OVX mice. Scale bar: upper panel, 100 μm; lower panel, 50 μm. Data are shown as mean ± SD ( n = 7 mice per group). * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using two-way ANOVA with Fisher’s LSD post hoc analysis. Source data are provided as a Source Data file.

Article Snippet: Wild-type BMSCs were differentiated in the mixed induction medium supplemented with 10% WAT-CM or BMAds-CM as indicated, in the presence of 0.5 μg/ml recombinant SPP1 protein (rSPP1, Abcam #ab281820), 1 μg/ml SPP1 neutralizing antibody (SPP1 Nab, Novus Biologicals #AF808), 0.5 μg/ml recombinant leptin protein (rLeptin, R&D Systems #490-OB-01M), 250 nM leptin receptor antagonist Allo-aca (MCE #HY-P3212) or an equal volume of vehicle IgG (Cell Signaling Technology #2729).

Techniques: Staining, Micro-CT, Labeling, Immunofluorescence

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Protein expression levels of ESRRA were evaluated in BMSCs upon adipogenic induction for indicated days, comparing Esrra fl/fl mice (blue font) with Esrra AKO mice (red font). b Schematic representation of the experimental design. BMSCs were isolated from Esrra fl/fl and Esrra AKO mice and subsequently subjected to either adipogenic or osteogenic induction for indicated days (schematic created with BioRender.com. Agreement number: LW26KBAHRA). c Representative images and quantitative analyses of alizarin red S staining and oil red O staining following the indicated induction. n = 4 biologically independent experiments. Rosi, rosiglitazone. d Volcano plot of transcriptional profiling between BMSCs-derived BMAds lineage cells from Esrra fl/fl and Esrra AKO mice. Differentially expressed genes were identified using DESeq2 analysis ( p < 0.05). n = 4 biologically independent samples. Gene Ontology (GO) ( e ) and Kyoto Encyclopedia of Genes and Genomes (KEGG) ( f ) pathway enrichment analyses of all differentially expressed genes by RNA-seq (top 10 according to adjusted p value). g Heatmap depicting selected genes related to secreted factors ( p < 0.05). n = 4 biologically independent samples. h Boxplot showing the transcript expression value (FPKM) of Spp1 based on RNA-seq data. Data are represented as box and whiskers with bars representing maximum and minimum values and with median highlighted as a line. n = 4 biologically independent samples. Validation of SPP1 and leptin expression were performed by qRT-PCR ( i ) and western blotting analysis ( j ) in BMAds that were fully differentiated for 14 days. n = 6 biologically independent samples. All the data are shown as mean ± SD. ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

Article Snippet: Wild-type BMSCs were differentiated in the mixed induction medium supplemented with 10% WAT-CM or BMAds-CM as indicated, in the presence of 0.5 μg/ml recombinant SPP1 protein (rSPP1, Abcam #ab281820), 1 μg/ml SPP1 neutralizing antibody (SPP1 Nab, Novus Biologicals #AF808), 0.5 μg/ml recombinant leptin protein (rLeptin, R&D Systems #490-OB-01M), 250 nM leptin receptor antagonist Allo-aca (MCE #HY-P3212) or an equal volume of vehicle IgG (Cell Signaling Technology #2729).

Techniques: Expressing, Isolation, Staining, Derivative Assay, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Plasma SPP1 levels of Esrra fl/fl and Esrra AKO mice at 8 weeks post-OVX or sham operation. n = 7 mice per group. b Representative images and analysis of SPP1 and leptin co-staining of gWAT from OVX mice studied in ( a ) (scale bar: 100 μm). n = 7 mice per group. c mRNA expression of Spp1 , Leptin , Adipoq , Pparg , Cebpa and Fabp4 of gWAT from OVX mice. n = 7 mice per group. d Protein levels of SPP1, leptin and ESRRA of gWAT from Esrra fl/fl and Esrra AKO mice at 4 and 8 weeks post-OVX or sham operation. e Schematic diagram displays the potential binding sites of ESR1 within the Spp1 promoter, including S1, S2 and S3. Fragments for ChIP assay shown as region 1 (R1) and region 2 (R2). f Luciferase reporter activities of the Spp1 promoter in adipogenesis induced 3T3-L1 cells transfected with Esrra or Esr1 expressing plasmids in the presence of E2 or not. n = 3 biologically independent experiments. The consensus sequence binding motifs for ESR1 response element (ERE) and ESRRA response element (ERRE) are presented. g Luciferase reporter activities of the Spp1 promoter regulated by E2/ESR1 in the presence of wild-type (WT) or DNA-binding domain-deleted ESRRA construct (ESRRA-ΔDBD). n = 4 biologically independent experiments. h ChIP assay with ESR1 antibody in BMSCs from Esrra fl/fl and Esrra AKO mice after adipogenic induction for 4 days along with or without E2. n = 3 biologically independent experiments. i Luciferase reporter activities of the R2 deleted- Spp1 promoter (ΔR2-luc) as compared to Spp1 promoter (WT-luc). n = 4 biologically independent experiments. j Enrichment of ESRRA in R2 of Spp1 promoter in adipogenesis induced 3T3-L1 cells with the indicated treatments. n = 3 biologically independent experiments. Spp1 mRNA in murine BMAds ( k ), matured 3T3-L1 adipocytes ( l ) or human BMSCs-derived BMAds ( m ) infected with adenovirus expressing ESRRA or GFP with E2 treatment for 2 days. n = 4, 6, 4 biologically independent experiments, respectively. n Diagram illustrating the mechanism of ESRRA-regulated repression of Spp1 transcriptional expression via interfering with E2/ESR1 signaling in adipocytes (schematic created with BioRender.com. Agreement number: BH26KF823M). Data are shown as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using unpaired two-tailed Student’s t test ( c ), one-way ANOVA followed by Bonferroni’s post hoc tests ( k , l , m ). Source data are provided as a Source Data file.

Article Snippet: Wild-type BMSCs were differentiated in the mixed induction medium supplemented with 10% WAT-CM or BMAds-CM as indicated, in the presence of 0.5 μg/ml recombinant SPP1 protein (rSPP1, Abcam #ab281820), 1 μg/ml SPP1 neutralizing antibody (SPP1 Nab, Novus Biologicals #AF808), 0.5 μg/ml recombinant leptin protein (rLeptin, R&D Systems #490-OB-01M), 250 nM leptin receptor antagonist Allo-aca (MCE #HY-P3212) or an equal volume of vehicle IgG (Cell Signaling Technology #2729).

Techniques: Staining, Expressing, Binding Assay, Luciferase, Transfection, Sequencing, Construct, Derivative Assay, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Representative images of metaphyseal type H vessels near growth plate immunostained for Endomucin (EMCN, red) and CD31 (green) in distal femurs of Esrra fl/fl and Esrra AKO mice following sham and OVX. DAPI (blue) is used for counterstaining of nuclei (scale bar: 100 μm). b Quantification of CD31 + EMCN + type H vessel intensity per mm 2 . n = 7 mice per group. c Immunostaining of Osterix (red) with DAPI (blue) in the metaphysis of distal femurs of Esrra fl/fl and Esrra AKO mice following sham and OVX (scale bar: 50 μm). d Quantification of Osterix + cells in bone marrow per mm 2 . n = 7 mice per group. e Schematic diagram showing the procedure of the conditioned medium (CM) preparation, tube formation assay and cell migration assay (schematic created with BioRender.com. Agreement number: VY26KB8O73). The concentrations of soluble SPP1 in BMAds-CM ( f ) or gWAT-CM ( g ) prepared from Esrra fl/fl and Esrra AKO OVX mice were measured by ELISA. n = 6, 4 biologically independent samples, respectively. Microvascular endothelial cells (ECs) migration in response to BMAds-CM ( h ) or gWAT-CM ( i ) with the addition of 0.5 μg/ml recombinant SPP1 (rSPP1), 1 μg/ml neutralizing SPP1 antibody (SPP1 Nab), or an equal volume of IgG for 24 h was followed by quantification and presentation of representative images from four independent experiments (scale bars: 100 μm). n = 4 biologically independent experiments. j – m Matrigel tube formation assay was performed using ECs and BMAds-CM ( j ) or gWAT-CM ( l ) with the indicated treatments for 4 h (scale bar: 100 μm). The length of the tubes and the number of branch points ( k , m ) per field were analyzed. n = 4 biologically independent experiments. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using unpaired two-tailed Student’s t test ( f , g ) and two-way ANOVA with Fisher’s LSD post hoc analysis ( b , d , h , i , k , m ). Source data are provided as a Source Data file.

Article Snippet: Wild-type BMSCs were differentiated in the mixed induction medium supplemented with 10% WAT-CM or BMAds-CM as indicated, in the presence of 0.5 μg/ml recombinant SPP1 protein (rSPP1, Abcam #ab281820), 1 μg/ml SPP1 neutralizing antibody (SPP1 Nab, Novus Biologicals #AF808), 0.5 μg/ml recombinant leptin protein (rLeptin, R&D Systems #490-OB-01M), 250 nM leptin receptor antagonist Allo-aca (MCE #HY-P3212) or an equal volume of vehicle IgG (Cell Signaling Technology #2729).

Techniques: Immunostaining, Tube Formation Assay, Cell Migration Assay, Enzyme-linked Immunosorbent Assay, Migration, Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Schematic diagram showing the procedure of the conditioned medium (CM) preparation from cultured BMAds or minced gWAT; and wild-type BMSCs were differentiated in osteogenic/adipogenic mixed induction medium (OIM:AIM = 1:1) supplemented with the indicated CM (schematic created with BioRender.com. Agreement number: ZP26KB8SN4). b The concentrations of soluble leptin in gWAT-CM prepared from Esrra fl/fl and Esrra AKO OVX mice were measured by ELISA. n = 4 biologically independent samples. c mRNA levels of osteogenesis markers Runx2 , Sp7 , Bglap , as well as adipogenic markers Pparg , Cebpa , Fabp4 in wild-type BMSCs cultured with mixed induction medium and indicated gWAT-CM for 14 days. n = 4 biologically independent experiments. Representative images and quantification of alizarin red S staining ( d ) and oil red O staining ( e ) of BMSCs cultured as in ( c ) with an addition of gWAT-CM for 14 days, in the presence of rSPP1, SPP1 Nab, recombinant leptin (rLeptin), leptin receptor antagonist Allo-aca or IgG as indicated. n = 4 biologically independent experiments. Scale bar: 2 mm ( d ); scale bar: 100 μm ( e ). f The concentrations of soluble leptin in BMAds-CM as prepared from ( a ). n = 6 biologically independent samples. g mRNA levels of indicated genes in wild-type BMSCs cultured in mixed induction medium supplemented with the indicated BMAds-CM for 14 days. n = 6 biologically independent experiments. Representative images and quantification of alizarin red S staining ( h ) and oil red O staining ( i ) of BMSCs cultured as in ( g ) with the indicated treatments for 14 days. The experiments were conducted according to the procedure shown in ( a – e ). n = 4 biologically independent experiments. Scale bar: 2 mm ( h ); scale bar: 100 μm ( i ). Data are shown as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using unpaired two-tailed Student’s t test ( b , c , f , g ) and two-way ANOVA with Fisher’s LSD post hoc analysis ( d , h , e , i ). Source data are provid e d as a Source Data file.

Article Snippet: Wild-type BMSCs were differentiated in the mixed induction medium supplemented with 10% WAT-CM or BMAds-CM as indicated, in the presence of 0.5 μg/ml recombinant SPP1 protein (rSPP1, Abcam #ab281820), 1 μg/ml SPP1 neutralizing antibody (SPP1 Nab, Novus Biologicals #AF808), 0.5 μg/ml recombinant leptin protein (rLeptin, R&D Systems #490-OB-01M), 250 nM leptin receptor antagonist Allo-aca (MCE #HY-P3212) or an equal volume of vehicle IgG (Cell Signaling Technology #2729).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Experimental design. Wild-type BMSCs were isolated from C57BL/6 mice and differentiated into BMAds, and 3T3-L1 preadipocytes were cultured in adipogenic medium for 14 days. Mature adipocytes were subsequently treated with C29 for an additional 2 days (schematic created with BioRender.com. Agreement number: IH26KB8VKA). The mRNA and protein levels of leptin and SPP1 in mature 3T3-L1 adipocytes ( b , c ) or BMAds ( d , e ). In vitro experiments were repeated four times. f Schematic diagram showing the experimental design for pharmacological treatments in DIO mice. Seven-week-old C57BL/6 mice were fed either a NCD or HFD for 18 weeks, and received either vehicle or C29 (30 mg/kg/body weight) every day during the last 4 weeks (schematic created with BioRender.com. Agreement number: WH26KBA0SE). g Plasma leptin, TNFa and IL6 levels. h , i Representative micro-CT images and histomorphometry analysis of BV/TV, Tb.N, Tb.Th and Tb.Sp at the distal femurs. j Representative micro-CT images of middle-segment of cortical bone and histomorphometry analysis of cortical bone volume/tissue volume ratio (BV/TV) and cortical thickness (Ct.Th). k Representative images of TRAP-stained femoral sections (scale bar: 100 μm). Quantitative assessment of trabecular Oc.S/BS and Oc.N/BS based on TRAP-stained sections. Plasma CTX-1 ( l ) and P1NP ( m ) levels. n Representative images of H&E-stained femur sections (scale bar: 100 μm). Quantitative assessment of trabecular Ob.S/BS and Ob.N/BS based on H&E-stained sections. o Representative PLIN1 and SPP1 immunostaining in femoral sections. Scale bar: upper panel, 100 μm; lower panel, 50 μm. p Quantification of PLIN1 + adipocyte number and of SPP1 fluorescence intensity. Six mice per group were used in all animal experiments. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using one-way ANOVA followed by Bonferroni’s post hoc tests ( b – d ) and two-way ANOVA with Fisher’s LSD post hoc analysis ( g , i – n , p ). Source data are provided as a Source Data file.

Article Snippet: Wild-type BMSCs were differentiated in the mixed induction medium supplemented with 10% WAT-CM or BMAds-CM as indicated, in the presence of 0.5 μg/ml recombinant SPP1 protein (rSPP1, Abcam #ab281820), 1 μg/ml SPP1 neutralizing antibody (SPP1 Nab, Novus Biologicals #AF808), 0.5 μg/ml recombinant leptin protein (rLeptin, R&D Systems #490-OB-01M), 250 nM leptin receptor antagonist Allo-aca (MCE #HY-P3212) or an equal volume of vehicle IgG (Cell Signaling Technology #2729).

Techniques: Isolation, Cell Culture, In Vitro, Micro-CT, Staining, Immunostaining, Fluorescence

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: Estrogen deficiency or high-fat diet-induced obesity results in excessive bone marrow adipocytes and distorted type H vessel. Adipocyte ESRRA deficiency preserves bone formation and counteracts high marrow adiposity by decreased leptin and enhanced SPP1 secretion, dictating BMSCs fate commitment toward osteogenesis and promoting vessel formation (schematic created with BioRender.com. Agreement number: QL26KB8YYQ).

Article Snippet: Wild-type BMSCs were differentiated in the mixed induction medium supplemented with 10% WAT-CM or BMAds-CM as indicated, in the presence of 0.5 μg/ml recombinant SPP1 protein (rSPP1, Abcam #ab281820), 1 μg/ml SPP1 neutralizing antibody (SPP1 Nab, Novus Biologicals #AF808), 0.5 μg/ml recombinant leptin protein (rLeptin, R&D Systems #490-OB-01M), 250 nM leptin receptor antagonist Allo-aca (MCE #HY-P3212) or an equal volume of vehicle IgG (Cell Signaling Technology #2729).

Techniques: