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: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Clinical Proteomics, 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: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Staining, Clinical Proteomics, 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: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Expressing, Isolation, Staining, Derivative Assay, RNA Sequencing, Biomarker Discovery, 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: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Clinical Proteomics, 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 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: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

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: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Isolation, Cell Culture, In Vitro, Clinical Proteomics, 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: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques:

Journal: PLoS ONE

Article Title: A Predictive Model of the Dynamics of Body Weight and Food Intake in Rats Submitted to Caloric Restrictions

doi: 10.1371/journal.pone.0100073

Figure Lengend Snippet: Plasma hormones and glucose assays.

Article Snippet: Plasma ghrelin and leptin assays were performed using immunoassays (acylated rat/mouse ghrelin #A05117 and rat/mouse leptin EIA #A05176, Cayman, SpiBio, Montigny le Bretonneux, France) according to the manufacturer’s recommendations.

Techniques: Clinical Proteomics

Journal: PLoS ONE

Article Title: A Predictive Model of the Dynamics of Body Weight and Food Intake in Rats Submitted to Caloric Restrictions

doi: 10.1371/journal.pone.0100073

Figure Lengend Snippet: Model variables.

Article Snippet: Plasma ghrelin and leptin assays were performed using immunoassays (acylated rat/mouse ghrelin #A05117 and rat/mouse leptin EIA #A05176, Cayman, SpiBio, Montigny le Bretonneux, France) according to the manufacturer’s recommendations.

Techniques: Clinical Proteomics