mouse rankl  (R&D Systems)

Journal: Bone Research

Article Title: Spatially resolved osteoblast-traced transcriptomics uncovers TGF-β as a combination target with sclerostin in osteoporosis

doi: 10.1038/s41413-026-00521-9

Figure Lengend Snippet: Dual inhibition of TGF-β and sclerostin increases bone mass in a hindlimb unloading model. a Experimental timeline of hindlimb unloading using C57B1/6 J mice and treatment with TGFβ-Ab, Scl-Ab, or both. Mice euthanized at week 12. b Representative μCT images of femoral diaphysis. c Representative confocal images of trabecular bone showing calcein (green), alizarin (red) signals, and DAPI (blue). Scale bar = 100 μm. d Representative images of TRAP-stained femur sections; violet = TRAP-positive, green = counterstain. Scale bar = 200 μm. e – h Bar plot of trabecular bone volume/total volume (Tra. BV/TV, e ), trabecular thickness (Tb. Th, f ), trabecular separation (Tb. Sp, g ), and trabecular number (Tb. N, h ) from μCT. Data = mean ± standard deviation. i , j Bar plot of mineral apposition rate (Tra. MAR, i ) and trabecular bone formation rate per bone surface (Tra. BFR/BS, j ) from labeling images. Data = mean ± standard deviation k Box plot of TRAP+ osteoclasts per bone surface (OC.N/BS), measured through ImageJ from TRAP-stained slides. l – n Box plot of serum P1NP ( l ) and TRAP ( m ) levels, and RANKL/OPG ratio ( n ). Group sizes: Control ( n = 5), Un_con ( n = 5), Scl-Ab ( n = 6), TGFβ-Ab ( n = 6), and Scl-Ab+TGFβ-Ab ( n = 7). Each point represents an individual mouse. ANOVA was used for statistics; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; ns not significant

Article Snippet: Serum levels of P1NP, CTx1, TRAcP, OPG, RANKL, and TGF-β1 were measured via an enzyme-linked immunosorbent assay (ELISA) performed using ELISA kit for mouse P1NP (AC-33F1, Immunodiagnostic Systems, UK), mouse CTx1 (AC-06F1, Immunodiagnostic Systems, UK), mouse TRAcP 5b (SB-TR103, Immunodiagnostic Systems, UK), mouse OPG (MOP00, R&D systems, USA), mouse RANKL (MTR00, R&D systems, USA), and mouse TGF-β1 (DY1679-05 and DY007B, R&D systems, USA) according to the manufacturer’s instruction.

Techniques: Inhibition, Staining, Standard Deviation, Labeling, Control

Journal: Molecular Medicine Reports

Article Title: Effects of zinc carnosine on bone loss in mice with diabetic osteoporosis

doi: 10.3892/mmr.2025.13723

Figure Lengend Snippet: COL-I, OCN, OPG and RANKL protein expression levels in each group after 8 weeks of intervention. (A) COL-I, OCN, OPG and RANKL protein expression level of each treatment group. Average optical density of (B) COL-I, (C) OCN, (D) OPG and (E) RANKL in each group. *P<0.05 vs. Ctrl group; # P<0.05 vs. T2DM model group. COL-I, type I collagen; OCN, osteocalcin; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand; Ctrl, control; T2DM, type 2 diabetes mellitus; ZnC, zinc carnosine; AOD, average optical density value.

Article Snippet: The primary antibodies used and their dilution ratios are as follows: Type I collagen (COL-I; 1:200; cat. no. AF7001; Affinity Biosciences), osteocalcin (OCN, 1:200; cat. no. 16157-1-AP; Proteintech Group, Inc.) and OPG (1:200; cat. no 31766-1-AP; Proteintech Group, Inc.).

Techniques: Expressing, Control

Journal: Molecular Medicine Reports

Article Title: Effects of zinc carnosine on bone loss in mice with diabetic osteoporosis

doi: 10.3892/mmr.2025.13723

Figure Lengend Snippet: mRNA expression levels of bone metabolism-related factors. *P<0.05 and ***P<0.01 vs. Ctrl group; ## P<0.01 vs. T2DM model group. (A) Ctrl, control; (B) T2DM, type 2 diabetes mellitus; (C) ZnC, zinc carnosine. OCN, osteocalcin; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand.

Article Snippet: The primary antibodies used and their dilution ratios are as follows: Type I collagen (COL-I; 1:200; cat. no. AF7001; Affinity Biosciences), osteocalcin (OCN, 1:200; cat. no. 16157-1-AP; Proteintech Group, Inc.) and OPG (1:200; cat. no 31766-1-AP; Proteintech Group, Inc.).

Techniques: Expressing, Control

Journal: JBMR Plus

Article Title: Neutralization of Receptor activator of nuclear factor-κB ligand reduces fibrosis and promotes osteoblast differentiation in a mouse model of fibrous dysplasia driven by somatic expression of Gnas R201H

doi: 10.1093/jbmrpl/ziaf145

Figure Lengend Snippet: Schematic depicting experiments utilizing the Sox9 CreERT ;Gnas R201H mouse model of FD. (A) Dams from crosses able to generate Sox9 CreERT ;Gnas (R201H)fl/+ ;Rosa26 LSL-tdTomato offspring were treated with a low dose of tamoxifen (40 mg/kg) at P6. Tamoxifen is then delivered to the pups via lactation, which allows expression of both mutant Gnas R201H and tdTomato as a lineage tracer in the subset of osteoprogenitor cells expressing Sox9 at P6. Resultant Gnas R201H ;R26 Tom mutant mice were then injected with either IgG2a or anti-RANKL antibody weekly until either P21 or P42 (3 or 6 wk of age). Femurs were collected for analysis. (B) Dams from crosses able to generate Sox9 CreERT ;Gnas (R201H)fl/+ ;Rosa26 LSL-tdTomato mice with either Rankl fl/fl or Rankl fl/+ or Rankl +/+ were genotyped to determine RANKL deletion. Pups were ear notched and genotyped at P5, the dam was treated with a low dose of tamoxifen (40 mg/kg) at P6 to deliver tamoxifen to the pups via lactation. Femurs were collected for analysis at either P21 or P42 (3 or 6 wk of age).

Article Snippet: ELISA was performed on serum diluted 2-fold with Calibrator Diluent RD6-12 using mouse RANKL immunoassay kit (MTR00; R&D System) according to the manufacturer’s directions.

Techniques: Expressing, Mutagenesis, Injection

Journal: JBMR Plus

Article Title: Neutralization of Receptor activator of nuclear factor-κB ligand reduces fibrosis and promotes osteoblast differentiation in a mouse model of fibrous dysplasia driven by somatic expression of Gnas R201H

doi: 10.1093/jbmrpl/ziaf145

Figure Lengend Snippet: Anti-RANKL treatment decreased osteoclasts and increases trabecular bone in the Gnas R201H ;R26 Tom mouse model of fibrous dysplasia. (A) 2D and 3D images of representative micro-CT scanned femurs show the bone architecture of 3-wk-old control Gnas + ;R26 Tom (left) and Gnas R201H ;R26 Tom mutant mice (right) treated with either anti-RANKL (aRANKL) or IgG2a isotype. Arrow denotes cortical irregularity. (B) Representative images of TRAP and fast green stained femur sections from Gnas R201H ;R26 Tom mice treated with aRANKL and IgG2a. (C) Quantification of trabecular bone area (BA/TA (%) from histologic sections. (D-H) A region of interest (ROI) extending 500 μm below the growth plate from IgG2a and aRANKL treated Gnas R201H ;R26 Tom mutant mice was analyzed for number of osteoclasts per bone surface (OC.N/BS (/mm)) and osteoclast surface per bone surface (OC.S/BS (%)) at either 3 wk (D-E) or 6 wk (F-I). (D) aRANKL reduced OC.N/BS and (E) OC.S/BS in 3-wk-old Gnas R201H ;R26 Tom mice. (F) Representative images of 6-wk-old IgG2a and aRANKL treated Gnas R201H ;R26 Tom mutant mice top, with higher magnification views of inset, bottom. (G) Quantification of the total tissue area of the ROI (T.Ar (mm 2 )). (H) The number of osteoclasts per total tissue area (OC.N/T.Ar (/mm 2 )). (I) The osteoclast surface per tissue area (OC.S/T.Ar (%)). Statistical analysis was performed using non-parametric Mann–Whitney t -test, where p < .05 was considered significant.

Article Snippet: ELISA was performed on serum diluted 2-fold with Calibrator Diluent RD6-12 using mouse RANKL immunoassay kit (MTR00; R&D System) according to the manufacturer’s directions.

Techniques: Micro-CT, Control, Mutagenesis, Staining, MANN-WHITNEY

Journal: JBMR Plus

Article Title: Neutralization of Receptor activator of nuclear factor-κB ligand reduces fibrosis and promotes osteoblast differentiation in a mouse model of fibrous dysplasia driven by somatic expression of Gnas R201H

doi: 10.1093/jbmrpl/ziaf145

Figure Lengend Snippet: Anti-RANKL treatment significantly reduced fibrosis in both 3- and 6-wk-old Gnas R201H ;R26 Tom FD mouse femurs. (A-C) Masson’s trichrome stained femur sections from 3-wk-old Gnas R201H ;R26 Tom mice treated with IgG2a isotype demonstrate fibrosis (arrows), which is absent in Sox9Cre ERT ;Gnas + ;R26 TOM WT mice (C) and reduced in aRANKL treated animals. (A and B) Fibrosis in the area below the growth plate and (D) in the femoral head. Scale bar = 200 μm (A, C), 100 μm (B). (E and F) Quantification of fibrosis in the femur ROI extending 500 μm below the growth plate from IgG2a and aRANKL treated 3-wk-old Gnas R201H ;R26 Tom and Gnas + ;R26 Tom control animals. (E) Fibrosis area per tissue area (Stromal.Ar/T.Ar (%)) and (F) tissue area (T.Ar (μm 2 )) for aRANKL and IgG2a treated 3-wk-old animals. (G and H) Masson’s trichrome stained femur sections from 6-wk-old Gnas R201H ;R26 Tom mice treated with IgG2a or aRANKL. Scale bar = 200 μm (G), 100 μm (H). (I and J) Fibrosis quantified in the femur ROI extending 500 μm below the growth plate in 6-wk-old Gnas R201H ;R26 Tom mice treated with IgG2a or aRANKL and Gnas + littermates. (I) Stromal.Ar/T.Ar (%) is reduced by aRANKL without changing (J) T.Ar (μm 2 ). Statistical analysis was performed using non-parametric Mann–Whitney t -test, where p < .05 was considered significant.

Article Snippet: ELISA was performed on serum diluted 2-fold with Calibrator Diluent RD6-12 using mouse RANKL immunoassay kit (MTR00; R&D System) according to the manufacturer’s directions.

Techniques: Staining, Control, MANN-WHITNEY

Journal: JBMR Plus

Article Title: Neutralization of Receptor activator of nuclear factor-κB ligand reduces fibrosis and promotes osteoblast differentiation in a mouse model of fibrous dysplasia driven by somatic expression of Gnas R201H

doi: 10.1093/jbmrpl/ziaf145

Figure Lengend Snippet: Anti-RANKL treatment of Gnas R201H ;R26 Tom mice shifts both WT and mutant cells to a more mature osteoblast phenotype, demonstrated by increased OCN and decreased SMA expression. (A-F) Representative confocal images of femur sections from 3-wk-old Gnas R201H ;R26 Tom treated with IgG2a or aRANKL. (A) The proximal metaphyseal region of the femur from 3-wk-old IgG2a and aRANKL treated Gnas R201H ;R26 Tom mutant mice shows endogenous TdTomato (TOM + ) signal in Gnas R201H mutant cells with DAPI nuclear staining. Scale bar = 100 μm. (B and C) Cells in the ROI extending 500 μm below the growth plate were quantified to determine (B) TOM + cells per tissue area (TOM + /T.Ar (/μm 2 )) and (C) TOM + cells per trabecular bone area (TOM + /B.Ar (/μm 2 )) and TOM + cells per bone marrow area (TOM + /bone marrow area (/μm 2 )) from IgG2a and aRANKL treated Gnas R201H ;R26 Tom mice. (D-F) Images of Gnas R201H ;R26 Tom femur sections immunostained for SMA, OSX, or OCN, with DAPI nuclear stain. Endogenous tdTomato signal identifies Gnas R201H mutant cells. (G-L) Cells in the ROI extending 500 μm below the growth plate were quantified to determine (G). Percentage of TOM + Gnas R201H mutant cells that co-expressed SMA (SMA + TOM + /TOM + (%)); (H) Total SMA + cells in the ROI tissue area (SMA + /T.Ar (/μm 2 )); (I) Percentage of TOM + Gnas R201H mutant cells that co-expressed OSX (OSX + TOM + /TOM + (%)); (J) Total OSX + cells in the ROI tissue area (OSX + /T.Ar (/μm 2 )); (K) Percentage of TOM + Gnas R201H mutant cells that co-expressed OCN (OCN + TOM + /TOM + (%)); and (L) Total OCN + cells per ROI tissue area (OCN + /T.Ar (/μm 2 )). Statistical analysis was performed using non-parametric Mann–Whitney t -test, where p < .05 was considered significant.

Article Snippet: ELISA was performed on serum diluted 2-fold with Calibrator Diluent RD6-12 using mouse RANKL immunoassay kit (MTR00; R&D System) according to the manufacturer’s directions.

Techniques: Mutagenesis, Expressing, Staining, MANN-WHITNEY

Journal: JBMR Plus

Article Title: Neutralization of Receptor activator of nuclear factor-κB ligand reduces fibrosis and promotes osteoblast differentiation in a mouse model of fibrous dysplasia driven by somatic expression of Gnas R201H

doi: 10.1093/jbmrpl/ziaf145

Figure Lengend Snippet: Rankl deletion in Gnas R201H ;R26 Tom mutant cells does not reduce fibrosis, alter SMA expression, nor reduce osteoclast formation in 6-wk-old mice. Formation of fibrotic lesions in Gnas R201H ;Rankl + ;R26 Tom and Gnas R201H ;Rankl Δ ;R26 Tom femurs was assessed by histology at (A-E, M-O) 3-wk and (F-L) 6-wk of age. Analysis was performed on the ROI 500 μm below the growth plate (A-L) or restricted to lesional area (M-O). (A) Representative images of TRAP stained femur sections from 3-wk-old animals counter stained with fast green. Scale bar = 200 μm. (B) Number of osteoclasts per bone surface (OC.N/BS (/mm)) and (C) osteoclast surface per bone surface (OC.S/BS (%)) within the ROI. (D) Representative images of Masson’s trichrome stained proximal metaphyseal region of the femur showed fibrosis (arrows) below the growth plate in both genotypes. Scale bar = 200 μm. (E) Fibrosis area per tissue area (Stromal.Ar/T.Ar (%)) for Gnas R201H ;Rankl Δ ;R26 Tom and Gnas R201H ;Rankl + ;R26 Tom mice. (F) Representative images of TRAP stained (pink) femur sections from 6-wk-old animals counter stained with fast green. Scale bar = 200 μm. (G) Number of osteoclasts per bone surface (OC.N/BS (/mm)), and (H) osteoclast surface per bone surface (OC.S/BS (%)) within the ROI. (I and J) Representative images of Masson’s trichrome stained proximal metaphyseal region of the femur showed fibrosis below the growth plate in both genotypes. Scale bar = 200 μm (I) and 100 μm (J). (K) Fibrosis area per tissue area (Stromal.Ar/T.Ar (%)), and (L) total tissue area (Stromal.Ar./T.AR(%)) for Gnas R201H ;Rankl Δ ;R26 Tom and Gnas R201H ;Rankl + ;R26 Tom mice, compared to Gnas + and Cre negative control littermates. (M) Representative images of SMA immunostained femur sections of 3-wk-old Gnas R201H ;Rankl + ;R26 Tom and Gnas R201H ;Rankl Δ ;R26 Tom mice. Scale bar = 50 μm. (N) Percentage of TOM + Gnas R201H mutant cells that co-expressed SMA within the lesion (SMA + TOM + /TOM + (%)). (O) Total SMA + cells per total cells (SMA + /DAPI + (%)), SMA + WT cells per total cells (SMA + TOM − /DAPI + (%)), and SMA + mutant cells per total cells (SMA + TOM + /DAPI + (%)). Statistical analysis of osteoclast parameters at both ages was performed using non-parametric Mann–Whitney t -test. Analysis of fibrosis was performed using an unpaired Student’s t -test for 3-wk-old animals and a one-way ANOVA with Tukey’s post-test for 6-wk-old animals. Analysis of SMA + cells was performed using an unpaired Student’s t -test. For all tests p < .05 was considered significant.

Article Snippet: ELISA was performed on serum diluted 2-fold with Calibrator Diluent RD6-12 using mouse RANKL immunoassay kit (MTR00; R&D System) according to the manufacturer’s directions.

Techniques: Mutagenesis, Expressing, Staining, Negative Control, MANN-WHITNEY

Journal: Scientific Reports

Article Title: A single acute alcohol intoxication before fracture insult causes long-term elevated systemic RANKL and OPG levels in young adult mice

doi: 10.1038/s41598-025-09240-3

Figure Lengend Snippet: Systemically analyses of bone turnover markers via ELISA. ( a ) Aged drunken mice with an isolated fracture showed a higher concentration of β-CTx in the plasma than the sober counterpart. ( b ) The RANKL value was increased in both older Fx and THFx mice after alcohol intoxication in contrast to the NaCl groups. ( c ) OPG only rose in drunken Fx animals in comparison to the not drunken ones. ( d ) For the RANKL/OPG ratio, young mice with isolated fracture with and without severe bleeding had higher values than the sober animals. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. indicated group; # p < 0.05 vs. respective Sham group. n = 5 per group.

Article Snippet: Analyses were made for β-CTx (#E-EL-M0372, Elabscience, Texas, USA), RANKL (#MTR00, R&D Systems, Minneapolis, USA) and OPG (#MOP00, R&D Systems) according to the manufacturers manuals.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Concentration Assay, Clinical Proteomics, Comparison