mouse rankl immunoassay kit  (R&D Systems)

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: Aging Cell

Article Title: Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

doi: 10.1111/acel.12586

Figure Lengend Snippet: Increased osteocyte apoptosis with reduced Cx43 and miR21 leads to increased RANKL / OPG ratio. (A) Expression of the indicated genes and in MLO ‐Y4 cells expressing or lacking Cx43 treated with vehicle or DEVD for 1 h and measured 24 h later. Bars represent mean ± SD ( n = 6). * P < 0.05 vs. corresponding sh RNA scramble cells and # P < 0.05 vs. vehicle‐treated Cx43 sh RNA cells by two‐way ANOVA . (B) mRNA levels for the indicated genes in calvaria from miR21 fl/fl mice treated with adenovirus‐ GFP or adenovirus‐Cre ( n = 3). Bars represent mean ± SD ( n = 3). * P < 0.05 vs. adenovirus‐ GFP ‐treated bone, by t ‐test. (C) HMGB 1 protein expression corrected by β‐actin measured in cell lysate and conditioned medium of MLO ‐Y4 osteocytic cells by Western blotting ( n = 3). (D,E) mRNA levels for RANKL and concentration of sRANKL and HMGB 1 in conditioned media from MLO ‐Y4 cells expressing or not Cx43, and treated with vehicle, DEVD or GA were measured by qPCR and ELISA , respectively ( n = 3). (F) Expression of the indicated genes corrected by GAPDH in MLO ‐Y4 cells expressing or not Cx43 and treated with nonimmune (ni) IgG or neutralizing anti‐ HMGB 1 antibodies for 24 h ( n = 6). * P < 0.05 vs. corresponding sh RNA scramble cells and # P < 0.05 vs. ni IgG‐treated Cx43 sh RNA cells by two‐way ANOVA.

Article Snippet: RANKL, OPG, and HMGB1 protein levels in the supernatants were determined using Quantikine Mouse RANKL Immunoassay (R&D Systems, Inc., Minneapolis, MN, USA, cat.# MTR00), Quantikine Osteoprotegerin Immusoassay (R&D Systems, Inc., cat.# MOP00), or HMGB1 Immunoassay (IBL International, Hamburg, Germany, cat.# ST51011), respectively.

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Aging Cell

Article Title: Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

doi: 10.1111/acel.12586

Figure Lengend Snippet: Blockage of apoptosis in Cx43‐silenced MLO ‐Y4 osteocytic cells reduces their osteoclastogenic potential. (A) Bone marrow cells isolated from C57Bl/6 mice and co‐cultured with MLO ‐Y4 osteocytic cells expressing or not Cx43 for 7 days. Multinucleated (≥ 3 nuclei) osteoclasts were enumerated. Bars represent mean ± SD ( n = 4). * P < 0.05 vs. sh RNA scramble cells by t ‐test. (B,C) Bone marrow cells were cultured in the presence of conditioned media from MLO ‐Y4 osteocytic cells expressing or not Cx43 and treated for 1 h with vehicle or DEVD . (B) Multinucleated osteoclasts and representative images of cultured osteoclasts stained by TRAP and (C) mRNA expression for the indicated genes assessed by qPCR were measured after 7‐day culture. Bars represent mean ± SD ( n = 4). * P < 0.05 vs. vehicle‐treated sh RNA scramble cells, and # P < 0.05 vs. vehicle‐treated Cx43 sh RNA cells, by two‐way ANOVA . Scale bars indicate 200 μm. (D) Multinucleated osteoclasts were enumerated after treatment of nonadherent bone marrow cells with conditioned media from MLO ‐Y4 osteocytic cells expressing or not Cx43 in the presence of vehicle or boxA. Bars represent mean ± SD ( n = 4). * P < 0.05 vs. corresponding sh RNA scramble cells, by two‐way ANOVA . Representative images of cultured osteoclasts stained by TRAP are shown. Scale bars indicate 100 μm. (E) Working model showing decreased osteocytic Cx43 leads to a reduction in miR21 levels which, in turn, leads to an increase in PTEN levels and consequently a reduction in Akt activation. Decreased Akt activity leads to increased expression of the apoptosis‐related genes p27, FoxO3, and GADD 153 resulting in caspase‐3‐mediated apoptosis. At the same time, apoptotic osteocytes release RANKL and HMGB 1, which induce osteoclast differentiation and recruitment.

Article Snippet: RANKL, OPG, and HMGB1 protein levels in the supernatants were determined using Quantikine Mouse RANKL Immunoassay (R&D Systems, Inc., Minneapolis, MN, USA, cat.# MTR00), Quantikine Osteoprotegerin Immusoassay (R&D Systems, Inc., cat.# MOP00), or HMGB1 Immunoassay (IBL International, Hamburg, Germany, cat.# ST51011), respectively.

Techniques: Isolation, Cell Culture, Expressing, Staining, Activation Assay, Activity Assay