cfd  (R&D Systems)

Journal: eLife

Article Title: Adipsin promotes bone marrow adiposity by priming mesenchymal stem cells

doi: 10.7554/eLife.69209

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Factor D Protein, Mouse, Recombinant (His Tag) , Sino Biological , 50539-M08H , 1 μg/mL.

Techniques: Recombinant, Staining, Electron Microscopy, Software

Journal: Bone Research

Article Title: Targeting adipocyte ESRRA alleviates osteoarthritis via interrupting inter-organelle crosstalk of complement C3-CFD-MAC cascade

doi: 10.1038/s41413-026-00527-3

Figure Lengend Snippet: Reduced CFD from Esrra -ablated BMAds interrupts C3-CFD-MAC cascade-mediated mitochondrial dysfunction, senescence and catabolism in chondrocytes. a , b Representative images ( a ) and quantification ( b ) of MAC and pERK1/2 positive staining in cartilage regions of HFHC-fed or 25-month-old Esrra fl/fl and Esrra AKO mice. Scale bar, 100 μm. n = 6 mice. c Diagram depicting primary wild-type chondrocytes treated with the indicated conditioned medium (CM) collected from mBMAds, with or without supplementation of 1 μg/mL mouse recombinant C3 (mC3). d Soluble CFD concentrations from BMAds CM prepared as in ( c ) ( n = 6). e mRNA levels of degradative enzymes Mmp13 , Mmp3 , Mmp9 , Adamts4 , Adamts5 and senescent/proinflammatory markers P16 , P53 , P21 , IL6 in co-cultured primary chondrocytes as in ( c ) ( n = 5). f Protein levels of pERK1/2, ERK1/2, P21, MMP13 and COL2A1 in co-cultured primary chondrocytes as in ( c ). g , h Immunofluorescence staining of MAC, pERK1/2, MitoTracker Red, MitoSOX Red, γH2AX, P21 and Ki67, as well as SA-β-gal staining in co-cultured primary chondrocytes ( g ). Quantitative analysis results are shown in ( h ) ( n = 4). DAPI or Hoechst was used for nuclear counterstaining. Scale bar, 10 μm. i Oxygen Consumption Rate (OCR) were measured in co-cultured primary chondrocytes as in ( c ). Bar chart showing the results of mitochondrial basal and maximal respiratory capacity changes ( n = 9). Data are shown as mean ± SD. Statistical analysis is performed using two-sided unpaired Student’s t -test ( b , d ), two-way ANOVA with post-hoc Turkey’s multiple comparisons test ( e , h , i )

Article Snippet: ELISA assays were performed to detect CFD (R&D Systems #DY5430-05), C3 (Cloud-clone #SEA861Mu), and P1NP (Novus Biologicals #NBP2-76466).

Techniques: Staining, Recombinant, Cell Culture, Immunofluorescence

Journal: Bone Research

Article Title: Targeting adipocyte ESRRA alleviates osteoarthritis via interrupting inter-organelle crosstalk of complement C3-CFD-MAC cascade

doi: 10.1038/s41413-026-00527-3

Figure Lengend Snippet: Inhibition of adipocyte ESRRA/CFD signaling rescues chondrocyte from damage caused by excessive hepatocytes-derived C3 under metabolic stress conditions. a Boxplots depicting C3 gene expression levels in MASLD/MASH patients compared to healthy controls from European ( GSE135251 ) and American ( GSE24807 ) cohorts. Data are represented as box and whiskers with bars representing maximum and minimum values and with median highlighted as a line. b Immunoblot analysis of C3 expression in livers from female mice at various time points following OVX (left) and male mice across different ages (right). c Representative liver images of the livers from HFHC-fed or 25-month-old Esrra fl/fl and Esrra AKO mice as well as corresponding controls. d Protein levels of C3 in the livers as in ( c ). e ELISA analysis of serum C3 concentrations. n = 6 mice per group. f The expression and secretion of C3 protein in primary hepatocytes following treatment with 40 μmol/L cholesterol (Chol) or a mixture of 250 μmol/L oleic acid (OA)/125 μmol/L palmitic acid (PA) for 24 hours (n = 4). g Diagram illustrating the co-culture system involving mBMAds CM, mouse primary hepatocytes and chondrocytes. h Western blot analysis of pERK1/2, ERK1/2, P21, MMP13 and COL2A1 in co-cultured primary chondrocytes as in ( g ). i , j Representative images ( i ) and quantitative analysis ( j ) of MAC, pERK1/2, MitoTracker Red, MitoSOX Red, γH2AX, P21, Ki67 and SA-β-gal staining in chondrocytes as in ( g ) ( n = 4). DAPI or Hoechst was used for counterstaining of nuclei. Scale bar, 10 μm. Data are shown as mean ± SD. Statistical analysis is performed using two-sided unpaired Student’s t -test (right panel of a ), two-way ANOVA with post-hoc Turkey’s multiple comparisons test ( e , j ), one-way ANOVA followed by Bonferroni’s post hoc tests (left panel of a , f )

Article Snippet: ELISA assays were performed to detect CFD (R&D Systems #DY5430-05), C3 (Cloud-clone #SEA861Mu), and P1NP (Novus Biologicals #NBP2-76466).

Techniques: Inhibition, Derivative Assay, Gene Expression, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Cell Culture, Staining

Journal: Bone Research

Article Title: Targeting adipocyte ESRRA alleviates osteoarthritis via interrupting inter-organelle crosstalk of complement C3-CFD-MAC cascade

doi: 10.1038/s41413-026-00527-3

Figure Lengend Snippet: Suppressing CFD by Danicopan or andrographolide confers protection against cellular senescence and catabolism in human C28/I2 chondrocytes. a Scatter plot showing plasma CFD levels across the lifespan were analyzed using a public proteome dataset (see Method for details). RFU, relative fluorescent unit. b Illustration of the co-culture system involving C28/I2 chondrocytes and conditioned medium (CM) obtained from AP-treated human BMAds (hBMAds), with an additional treatment of 1 μg/mL human recombinant C3 (hC3) and/or 10 mmol/L Danicopan. c Western blot analysis of ESRRA, CFD and Leptin protein levels in human BMSCs-derived BMAds treated with 40 μmol/L AP or DMSO for 2 days. d Western blot analysis of pERK1/2, ERK1/2, P21, MMP13, and COL2A1 protein levels in human C28/I2 chondrocytes as in ( b ). e , f Representative images ( e ) and corresponding quantitative data ( f ) of MAC, pERK1/2, MitoTracker Red, MitoSOX Red, γH2AX, P21, Ki67 and SA-β-gal staining in human C28/I2 chondrocytes as in ( b ) ( n = 4). DAPI or Hoechst was used for nuclear counterstaining. Scale bar, 10 μm. Data are shown as mean ± SD. Statistical analysis is performed using two-way ANOVA with post-hoc Turkey’s multiple comparisons test

Article Snippet: ELISA assays were performed to detect CFD (R&D Systems #DY5430-05), C3 (Cloud-clone #SEA861Mu), and P1NP (Novus Biologicals #NBP2-76466).

Techniques: Clinical Proteomics, Co-Culture Assay, Recombinant, Western Blot, Derivative Assay, Staining

Journal: Bone Research

Article Title: Targeting adipocyte ESRRA alleviates osteoarthritis via interrupting inter-organelle crosstalk of complement C3-CFD-MAC cascade

doi: 10.1038/s41413-026-00527-3

Figure Lengend Snippet: Pharmacological inhibition of ESRRA by andrographolide protects aged mice against spontaneous osteoarthritis progression. a Schematic diagram of the experimental design for pharmacological treatments in mice. 22-month-old male C57BL/6 mice were administered intragastrically (i.g.) with either vehicle or AP (100 mg/kg body weight) five times per week for 3 months. Young controls were 3-month-old male mice. b Representative images of WAT depots and livers, along with WAT weight analysis. c Liver TC and TG levels. d Immunoblots of C3 in liver tissues. e Serum C3 levels. f Immunoblots of bone marrow CFD and Leptin. g Serum CFD levels. h , i Immunofluorescence of PLIN1 staining ( h ) and quantification of PLIN1 + bone marrow adipocytes area ( i ). Scale bar, 100 μm. j Representative SO&FG-stained joint sections and H&E-stained synovial tissue. Scale bar, 100 μm. k , l OARSI score ( k ) and synovitis score ( l ). m Digital microCT images of knee joints. n , Quantitative data of the volume of calcified meniscus and synovial tissue. o , p Immunofluorescence staining for MAC and pERK1/2 in articular cartilage ( o ) and the quantitation ( p ). Scale bar, 100 μm. q – t Immunofluorescence staining ( q , r ) and quantification ( s , t ) of p21, Ki67, MMP13 and COL2A1. Scale bar, 100 μm. u A proposed model illustrates that ESRRA-mediated adipocyte CFD and liver-derived C3 synergistically promote osteoarthritis progression in aging and metabolic disorders. ESRRA transcriptionally upregulates CFD responding to MAT expansion. Together with steatotic liver-derived C3, this triggers excessive alternative complement activation, resulting in MAC deposition on chondrocytes, therefore provoking pERK1/2 activation and mitochondrial dysfunction. Targeting ESRRA alleviates osteoarthritis by interrupting this inter-organ C3-CFD-MAC cascade. For all experiments, n = 6 mice. Data are shown as mean ± SD. Statistical analysis is performed using two-way ANOVA with post-hoc Turkey’s multiple comparisons test

Article Snippet: ELISA assays were performed to detect CFD (R&D Systems #DY5430-05), C3 (Cloud-clone #SEA861Mu), and P1NP (Novus Biologicals #NBP2-76466).

Techniques: Inhibition, Western Blot, Immunofluorescence, Staining, Quantitation Assay, Derivative Assay, Activation Assay

Journal: Bone Research

Article Title: Targeting adipocyte ESRRA alleviates osteoarthritis via interrupting inter-organelle crosstalk of complement C3-CFD-MAC cascade

doi: 10.1038/s41413-026-00527-3

Figure Lengend Snippet: Adipocyte ESRRA positively regulates Cfd transcriptional expression responding to bone marrow adipocytes expansion. a Venn diagram of overlapping differentially expressed genes from RNA-seq of HFHC-fed gWAT and BMAds lineage cells in Esrra fl/fl and Esrra AKO mice ( GSE248799 ), identifying six downregulated genes encoding secreted factors. b Heatmap of selected genes. c Cf d mRNA in HFHC-fed gWAT ( n = 6) and fully differentiated mBMAds ( n = 5). d Schematic of ERRE binding sites on the mouse Cfd promoter, with ChIP fragments indicated as region 1 and 2 (R1, R2). e Luciferase activity of Cfd promoter in 3T3‑L1 cells transfected with Esrra or Ppargc1a expression plasmids ( n = 5). f Effects of compound 29 (C29) or andrographolide (AP) on ESRRA/PPARGC1A- driven Cfd promoter activity ( n = 5). g Luciferase activities of the R2-deleted (ΔR2-luc), S2-mutated (MutS2-luc) and wild-type (WT-luc) Cfd promoter ( n = 5). h ChIP assays with ESRRA antibody or IgG control in BMSCs after 4 days of adipogenic induction, with cells infected with ESRRA- or GFP-expressing adenovirus ( n = 4). i Enrichment of ESRRA at Cfd promoter in BMSCs undergoing adipogenic induction from Esrra fl/fl and Esrra AKO mice ( n = 4). j , k Immunoblots of serum CFD from HFHC-fed ( j ) and aged ( k ) groups versus control diet (CD). Ponceau S as loading control (L.C). l Serum CFD levels ( n = 6). m , n Immunoblots of CFD and Leptin in gWAT from Esrra fl/fl and Esrra AKO mice as in ( j , k ). o CFD concentrations from culture medium of gWAT explants as in ( j , k ) ( n = 5). p , q Immunoblots of bone marrow CFD as in ( j , k ). r Protein levels of ESRRA, CFD and Leptin in BMSCs and mBMAds with treatment of dexamethasone (DEX) with or without rosiglitazone (Rosi). Data are shown as mean ± SD. Statistical analysis is performed using two-sided unpaired Student’s t -test ( c ), two-way ANOVA with post-hoc Turkey’s multiple comparisons test ( l , o )

Article Snippet: The antibodies used for immunoblot analysis included ESRRA (1:1 000, Cell Signaling Technology #13826), Tubulin (1:5 000, BPI #AbM59005-37B-PU), mouse CFD (1:800, R&D Systems #AF5430), human CFD (ABclonal #A23006), C3 (1:5 000, ABclonal #A13283), GAPDH (1:5 000, Proteintech #60004-1-Ig), COL2A1 (1:800, Proteintech #28459-1-AP), MMP13 (1:1 000, Proteintech #18165-1-AP), P21 (1:1 000, Proteintech #28248-1-AP), pERK1/2 (1:2 000, Abmart #T40072S), ERK1/2 (1:2 000, Abmart #T40071S), and Leptin (1:500, Abcam #ab16227).

Techniques: Expressing, RNA Sequencing, Binding Assay, Luciferase, Activity Assay, Transfection, Control, Infection, Western Blot

Journal: Bone Research

Article Title: Targeting adipocyte ESRRA alleviates osteoarthritis via interrupting inter-organelle crosstalk of complement C3-CFD-MAC cascade

doi: 10.1038/s41413-026-00527-3

Figure Lengend Snippet: Reduced CFD from Esrra -ablated BMAds interrupts C3-CFD-MAC cascade-mediated mitochondrial dysfunction, senescence and catabolism in chondrocytes. a , b Representative images ( a ) and quantification ( b ) of MAC and pERK1/2 positive staining in cartilage regions of HFHC-fed or 25-month-old Esrra fl/fl and Esrra AKO mice. Scale bar, 100 μm. n = 6 mice. c Diagram depicting primary wild-type chondrocytes treated with the indicated conditioned medium (CM) collected from mBMAds, with or without supplementation of 1 μg/mL mouse recombinant C3 (mC3). d Soluble CFD concentrations from BMAds CM prepared as in ( c ) ( n = 6). e mRNA levels of degradative enzymes Mmp13 , Mmp3 , Mmp9 , Adamts4 , Adamts5 and senescent/proinflammatory markers P16 , P53 , P21 , IL6 in co-cultured primary chondrocytes as in ( c ) ( n = 5). f Protein levels of pERK1/2, ERK1/2, P21, MMP13 and COL2A1 in co-cultured primary chondrocytes as in ( c ). g , h Immunofluorescence staining of MAC, pERK1/2, MitoTracker Red, MitoSOX Red, γH2AX, P21 and Ki67, as well as SA-β-gal staining in co-cultured primary chondrocytes ( g ). Quantitative analysis results are shown in ( h ) ( n = 4). DAPI or Hoechst was used for nuclear counterstaining. Scale bar, 10 μm. i Oxygen Consumption Rate (OCR) were measured in co-cultured primary chondrocytes as in ( c ). Bar chart showing the results of mitochondrial basal and maximal respiratory capacity changes ( n = 9). Data are shown as mean ± SD. Statistical analysis is performed using two-sided unpaired Student’s t -test ( b , d ), two-way ANOVA with post-hoc Turkey’s multiple comparisons test ( e , h , i )

Article Snippet: The antibodies used for immunoblot analysis included ESRRA (1:1 000, Cell Signaling Technology #13826), Tubulin (1:5 000, BPI #AbM59005-37B-PU), mouse CFD (1:800, R&D Systems #AF5430), human CFD (ABclonal #A23006), C3 (1:5 000, ABclonal #A13283), GAPDH (1:5 000, Proteintech #60004-1-Ig), COL2A1 (1:800, Proteintech #28459-1-AP), MMP13 (1:1 000, Proteintech #18165-1-AP), P21 (1:1 000, Proteintech #28248-1-AP), pERK1/2 (1:2 000, Abmart #T40072S), ERK1/2 (1:2 000, Abmart #T40071S), and Leptin (1:500, Abcam #ab16227).

Techniques: Staining, Recombinant, Cell Culture, Immunofluorescence

Journal: Bone Research

Article Title: Targeting adipocyte ESRRA alleviates osteoarthritis via interrupting inter-organelle crosstalk of complement C3-CFD-MAC cascade

doi: 10.1038/s41413-026-00527-3

Figure Lengend Snippet: Inhibition of adipocyte ESRRA/CFD signaling rescues chondrocyte from damage caused by excessive hepatocytes-derived C3 under metabolic stress conditions. a Boxplots depicting C3 gene expression levels in MASLD/MASH patients compared to healthy controls from European ( GSE135251 ) and American ( GSE24807 ) cohorts. Data are represented as box and whiskers with bars representing maximum and minimum values and with median highlighted as a line. b Immunoblot analysis of C3 expression in livers from female mice at various time points following OVX (left) and male mice across different ages (right). c Representative liver images of the livers from HFHC-fed or 25-month-old Esrra fl/fl and Esrra AKO mice as well as corresponding controls. d Protein levels of C3 in the livers as in ( c ). e ELISA analysis of serum C3 concentrations. n = 6 mice per group. f The expression and secretion of C3 protein in primary hepatocytes following treatment with 40 μmol/L cholesterol (Chol) or a mixture of 250 μmol/L oleic acid (OA)/125 μmol/L palmitic acid (PA) for 24 hours (n = 4). g Diagram illustrating the co-culture system involving mBMAds CM, mouse primary hepatocytes and chondrocytes. h Western blot analysis of pERK1/2, ERK1/2, P21, MMP13 and COL2A1 in co-cultured primary chondrocytes as in ( g ). i , j Representative images ( i ) and quantitative analysis ( j ) of MAC, pERK1/2, MitoTracker Red, MitoSOX Red, γH2AX, P21, Ki67 and SA-β-gal staining in chondrocytes as in ( g ) ( n = 4). DAPI or Hoechst was used for counterstaining of nuclei. Scale bar, 10 μm. Data are shown as mean ± SD. Statistical analysis is performed using two-sided unpaired Student’s t -test (right panel of a ), two-way ANOVA with post-hoc Turkey’s multiple comparisons test ( e , j ), one-way ANOVA followed by Bonferroni’s post hoc tests (left panel of a , f )

Article Snippet: The antibodies used for immunoblot analysis included ESRRA (1:1 000, Cell Signaling Technology #13826), Tubulin (1:5 000, BPI #AbM59005-37B-PU), mouse CFD (1:800, R&D Systems #AF5430), human CFD (ABclonal #A23006), C3 (1:5 000, ABclonal #A13283), GAPDH (1:5 000, Proteintech #60004-1-Ig), COL2A1 (1:800, Proteintech #28459-1-AP), MMP13 (1:1 000, Proteintech #18165-1-AP), P21 (1:1 000, Proteintech #28248-1-AP), pERK1/2 (1:2 000, Abmart #T40072S), ERK1/2 (1:2 000, Abmart #T40071S), and Leptin (1:500, Abcam #ab16227).

Techniques: Inhibition, Derivative Assay, Gene Expression, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Cell Culture, Staining

Journal: Bone Research

Article Title: Targeting adipocyte ESRRA alleviates osteoarthritis via interrupting inter-organelle crosstalk of complement C3-CFD-MAC cascade

doi: 10.1038/s41413-026-00527-3

Figure Lengend Snippet: Suppressing CFD by Danicopan or andrographolide confers protection against cellular senescence and catabolism in human C28/I2 chondrocytes. a Scatter plot showing plasma CFD levels across the lifespan were analyzed using a public proteome dataset (see Method for details). RFU, relative fluorescent unit. b Illustration of the co-culture system involving C28/I2 chondrocytes and conditioned medium (CM) obtained from AP-treated human BMAds (hBMAds), with an additional treatment of 1 μg/mL human recombinant C3 (hC3) and/or 10 mmol/L Danicopan. c Western blot analysis of ESRRA, CFD and Leptin protein levels in human BMSCs-derived BMAds treated with 40 μmol/L AP or DMSO for 2 days. d Western blot analysis of pERK1/2, ERK1/2, P21, MMP13, and COL2A1 protein levels in human C28/I2 chondrocytes as in ( b ). e , f Representative images ( e ) and corresponding quantitative data ( f ) of MAC, pERK1/2, MitoTracker Red, MitoSOX Red, γH2AX, P21, Ki67 and SA-β-gal staining in human C28/I2 chondrocytes as in ( b ) ( n = 4). DAPI or Hoechst was used for nuclear counterstaining. Scale bar, 10 μm. Data are shown as mean ± SD. Statistical analysis is performed using two-way ANOVA with post-hoc Turkey’s multiple comparisons test

Article Snippet: The antibodies used for immunoblot analysis included ESRRA (1:1 000, Cell Signaling Technology #13826), Tubulin (1:5 000, BPI #AbM59005-37B-PU), mouse CFD (1:800, R&D Systems #AF5430), human CFD (ABclonal #A23006), C3 (1:5 000, ABclonal #A13283), GAPDH (1:5 000, Proteintech #60004-1-Ig), COL2A1 (1:800, Proteintech #28459-1-AP), MMP13 (1:1 000, Proteintech #18165-1-AP), P21 (1:1 000, Proteintech #28248-1-AP), pERK1/2 (1:2 000, Abmart #T40072S), ERK1/2 (1:2 000, Abmart #T40071S), and Leptin (1:500, Abcam #ab16227).

Techniques: Clinical Proteomics, Co-Culture Assay, Recombinant, Western Blot, Derivative Assay, Staining

Journal: Bone Research

Article Title: Targeting adipocyte ESRRA alleviates osteoarthritis via interrupting inter-organelle crosstalk of complement C3-CFD-MAC cascade

doi: 10.1038/s41413-026-00527-3

Figure Lengend Snippet: Pharmacological inhibition of ESRRA by andrographolide protects aged mice against spontaneous osteoarthritis progression. a Schematic diagram of the experimental design for pharmacological treatments in mice. 22-month-old male C57BL/6 mice were administered intragastrically (i.g.) with either vehicle or AP (100 mg/kg body weight) five times per week for 3 months. Young controls were 3-month-old male mice. b Representative images of WAT depots and livers, along with WAT weight analysis. c Liver TC and TG levels. d Immunoblots of C3 in liver tissues. e Serum C3 levels. f Immunoblots of bone marrow CFD and Leptin. g Serum CFD levels. h , i Immunofluorescence of PLIN1 staining ( h ) and quantification of PLIN1 + bone marrow adipocytes area ( i ). Scale bar, 100 μm. j Representative SO&FG-stained joint sections and H&E-stained synovial tissue. Scale bar, 100 μm. k , l OARSI score ( k ) and synovitis score ( l ). m Digital microCT images of knee joints. n , Quantitative data of the volume of calcified meniscus and synovial tissue. o , p Immunofluorescence staining for MAC and pERK1/2 in articular cartilage ( o ) and the quantitation ( p ). Scale bar, 100 μm. q – t Immunofluorescence staining ( q , r ) and quantification ( s , t ) of p21, Ki67, MMP13 and COL2A1. Scale bar, 100 μm. u A proposed model illustrates that ESRRA-mediated adipocyte CFD and liver-derived C3 synergistically promote osteoarthritis progression in aging and metabolic disorders. ESRRA transcriptionally upregulates CFD responding to MAT expansion. Together with steatotic liver-derived C3, this triggers excessive alternative complement activation, resulting in MAC deposition on chondrocytes, therefore provoking pERK1/2 activation and mitochondrial dysfunction. Targeting ESRRA alleviates osteoarthritis by interrupting this inter-organ C3-CFD-MAC cascade. For all experiments, n = 6 mice. Data are shown as mean ± SD. Statistical analysis is performed using two-way ANOVA with post-hoc Turkey’s multiple comparisons test

Article Snippet: The antibodies used for immunoblot analysis included ESRRA (1:1 000, Cell Signaling Technology #13826), Tubulin (1:5 000, BPI #AbM59005-37B-PU), mouse CFD (1:800, R&D Systems #AF5430), human CFD (ABclonal #A23006), C3 (1:5 000, ABclonal #A13283), GAPDH (1:5 000, Proteintech #60004-1-Ig), COL2A1 (1:800, Proteintech #28459-1-AP), MMP13 (1:1 000, Proteintech #18165-1-AP), P21 (1:1 000, Proteintech #28248-1-AP), pERK1/2 (1:2 000, Abmart #T40072S), ERK1/2 (1:2 000, Abmart #T40071S), and Leptin (1:500, Abcam #ab16227).

Techniques: Inhibition, Western Blot, Immunofluorescence, Staining, Quantitation Assay, Derivative Assay, Activation Assay