Journal: Molecular medicine reports

Article Title: Release from optimal compressive force suppresses osteoclast differentiation.

doi: 10.3892/mmr.2016.5801

Figure Lengend Snippet: Figure 4. Effects of release from optimal CF on mRNA levels of osteoclast‑associated genes for 1‑24 h. mRNA levels of osteoclast‑associated genes (A) NFATc1 (B) TRAP (C) RANK (D) MMP‑9 (E) Cath‑K (F) ClC7 (G) ATP6i (H) DC‑STAMP and (I) OC‑STAMP were evaluated by reverse transcription‑quantitative polymerase reaction. Results are presented as the mean ± standard deviation (n=4). *P<0.05, **P<0.01, comparison indicated by brackets. cont, control; CF, compressive force; NFATc1, nuclear factor of activated T cells 1; TRAP, tartrate‑resistant acid phosphatase; RANK, receptor activator of nuclear factor‑κB; MMP‑9, matrix metalloproteinase‑9; Cath‑K, cathepsin‑K; ClC7, chloride channel 7; ATP6i, ATPase H+ transporting vacuolar proton pump member I; DC‑STAMP, dendritic cell‑specific transmembrane protein; OC‑STAMP, osteoclast stimulatory transmembrane protein.

Article Snippet: The following specific TaqMan probes (Applied Biosystems; Thermo Fisher Scientific, Inc.) for osteoclast‐associated genes were used: NFATc1 (ID no. Mm00479445_ml), TRAP (ID no. Mm00475698_ m l ) , m a t r i x m e t a l l o p r o t e i n a s e - 9 ( M M P - 9 ; ID no. Mm00432271_ml), RANK (ID no. Mm‐00437135_ml), osteoclast stimulatory trans membrane protein (OC-STAMP; ID no. Mm00512445_ml), dendritic cell specific trans membrane protein (DC-STAMP; ID no. Mm01168058_ml), cathepsin‐K (Cath‐K; ID no. Mm00484036_ml), chloride channel 7 (ClC-7; ID no. Mm00442400_ml) and ATPase H+ transporting vacuolar proton pump member I (ATP6i; ID no. Mm00469395_gl).

Techniques: Standard Deviation, Comparison, Control

Journal: Bioactive materials

Article Title: Calcium phosphate-based materials regulate osteoclast-mediated osseointegration.

doi: 10.1016/j.bioactmat.2021.05.003

Figure Lengend Snippet: Fig. 5. Effect of Ca/P ratio in CPC on RANKL-RANK signaling. (a) Immunofluorescence analysis of RANK expression (red) in osteoclasts on surface of CPC scaffold. (b) Concentration of RANK was analysis by ELISA. The concentration of RANK was measured after inducing BMMs for 1 days. (c) Affinity between RANKL and RANK was measured in BIAcore T200 system. Human-RANK anchored on the chip was applied to interact with the human-RANKL dissolved into the CPC extract. The vehicle means MEM-α medium. (d) 1.67CPC promoted RANKL-induced phosphorylation of NF-κB p65, and degradation of IκBα. (e) p-p65 and (f) IκBα intensity of Western blot bands was calculated with normalizing to β-actin. The untreated medium was treated as control. (g–l) Relative mRNA expression of osteoclast was measured after inducing BMMs for 7 days in CPC extract. BMMs cultured with untreated medium was treated as control. (*P ＜ 0.05, **P ＜ 0.01,***P ＜ 0.001).

Article Snippet: The concentration of RANK was measured by Mouse RANK ELISA Kit (Boster Biological Technology, China), according to the manufacturer’s instructions and the total protein concentration was measured by BCA Protein Assay Kit (Beyotime, China), which was used to normalize the expression of RANK.

Techniques: Immunofluorescence, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control, Cell Culture

Journal: Journal of Bone Oncology

Article Title: RANK and RANK ligand expression in primary human osteosarcoma

doi: 10.1016/j.jbo.2015.06.002

Figure Lengend Snippet: RANKL and RANK IHC of GCTB. IHC performed on FFPE section of GCTB, which serves as an internal positive and negative control since RANKL and RANK are expressed in distinct compartments . GCTB is composed of osteoclast-like giant cells and myeloid giant cell precursors that express RANK and stromal tumor cells that express RANKL. (A) The RANKL IHC using M366 clearly indicates stromal tumor cells but is excluded from the giant cell component. (B) Conversely, the RANK IHC using N-2B10 recognizes giant cells and myeloid osteoclast precursors but is excluded from the stromal compartment. FFPE, formalin-fixed, paraffin-embedded; GCTB, giant cell tumor of bone; IHC, immunohistochemistry; RANK, receptor activator of nuclear factor kappa-B; RANKL, RANK ligand.

Article Snippet: Ten nanograms of tumor cell cDNA was analyzed for RANK expression using Assay on DemandTM primer probe set Hs00187189_m1 (Applied Biosystems, Inc.).

Techniques: Negative Control, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: Journal of Bone Oncology

Article Title: RANK and RANK ligand expression in primary human osteosarcoma

doi: 10.1016/j.jbo.2015.06.002

Figure Lengend Snippet: huRANK antibody validation for IHC methods. (A) RANK IHC was performed on FFPE samples of xenografts of the specified human cancer cells. Parental cells did not detectably express RANK as confirmed by multiple independent methods. There was no detectable signal with the isotype control antibody. Both antibodies against huRANK (N-1H8 and N-2B10) provide a similar staining pattern, essentially cross-validating one another. (B) The same cells used for the above tumor xenografts were grown in vitro and processed for flow cytometry. For anti-RANK staining, the pattern of staining by both test antibodies N-1H8 and N-2B10 was compared with a previously identified mAb (M331) useful for flow cytometry applications (Armstrong ). Solid grey line : Unstained; Red line : Secondary control=Goat antimouse APC; Blue line : Isotype control 4D2 (anti-AGP3 muIgG1), 1 μg/mL; Purple line : M331 (anti-huRANK muIgG1), 1 μg/mL; Green line : N-1H8 (anti-huRANK muIgG1), 1 μg/mL; Black line : N-2B10 (anti-huRANK muIgG1), 1 μg/mL. The RANK signal by IHC is concordant with the signal by flow cytometry. APC, allophycocyanin; FFPE, formalin-fixed, paraffin-embedded; IHC, immunohistochemistry; mAb, monoclonal antibody; RANK, receptor activator of nuclear factor kappa-B.

Article Snippet: Ten nanograms of tumor cell cDNA was analyzed for RANK expression using Assay on DemandTM primer probe set Hs00187189_m1 (Applied Biosystems, Inc.).

Techniques: Biomarker Discovery, Control, Staining, In Vitro, Flow Cytometry, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: Journal of Bone Oncology

Article Title: RANK and RANK ligand expression in primary human osteosarcoma

doi: 10.1016/j.jbo.2015.06.002

Figure Lengend Snippet: RANKL and RANK H-score distribution in OS. The H score incorporates intensity (scale of 0–3) and percentage of tumor cells stained positive, giving a range of 0–300. RANK IHC was performed using N-1H8 mAb on the small core TMA samples and confirmed using N-2B10 mAb on the larger OS tumor sections. RANKL IHC was performed using M366 mAb on both the TMA and large tissue sections. The H-score distribution for both analytes are depicted for both (A) the small TMA core samples and (B) the larger tissue sections. IHC, immunohistochemistry; OS, osteosarcoma; RANK, receptor activator of nuclear factor kappa-B; RANKL, RANK ligand.

Article Snippet: Ten nanograms of tumor cell cDNA was analyzed for RANK expression using Assay on DemandTM primer probe set Hs00187189_m1 (Applied Biosystems, Inc.).

Techniques: Staining, Immunohistochemistry

Journal: Journal of Bone Oncology

Article Title: RANK and RANK ligand expression in primary human osteosarcoma

doi: 10.1016/j.jbo.2015.06.002

Figure Lengend Snippet: Range of RANKL protein expression in OS. RANKL IHC performed with mAb M366. Representative IHC images from four separate OS tumor samples are shown. Arrows indicate RANKL-positive tumor cells and tumor anaplastic cells. In addition, RANKL-negative osteoclasts are also clearly delineated (arrow). The four images demonstrate a range of RANKL positivity, as indicated by the different H-scores. The H-score for each image was as follows: upper left panel, H-score=260; upper right panel, H-score=210; lower left panel, H-score=180; lower right panel, H-score=20. IHC, immunohistochemistry; OS, osteosarcoma; RANK, receptor activator of nuclear factor kappa-B ligand.

Article Snippet: Ten nanograms of tumor cell cDNA was analyzed for RANK expression using Assay on DemandTM primer probe set Hs00187189_m1 (Applied Biosystems, Inc.).

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Bone Oncology

Article Title: RANK and RANK ligand expression in primary human osteosarcoma

doi: 10.1016/j.jbo.2015.06.002

Figure Lengend Snippet: RANKL and RANK expression within adjacent bone tissue. RANKL and RANK IHC performed with mAb N-1H8 and M366, respectively. (A) RANKL-expressing stroma at bone interface. (B) RANK expression in bone osteoclast. IHC, immunohistochemistry; mAb, monoclonal antibody; RANK, receptor activator of nuclear factor kappa-B; RANKL, RANK ligand.

Article Snippet: Ten nanograms of tumor cell cDNA was analyzed for RANK expression using Assay on DemandTM primer probe set Hs00187189_m1 (Applied Biosystems, Inc.).

Techniques: Expressing, Immunohistochemistry

Journal: PLoS ONE

Article Title: Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors

doi: 10.1371/journal.pone.0048795

Figure Lengend Snippet: BMMs over-expressing RANK (transduced with pMX-RANK-puro vector) or TRAF6 (transduced with pMX-TRAF6-puro vector) were cultured with M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) for 4 days under the adherent or non-adherent condition, then fixed and stained for TRAP. Cell under non-adherent condition were harvested and placed on cell culture plates for 1 hour to stain for TRAP.

Article Snippet: RANK, NFATc1, and integrin β 3 mRNA expressions were quantified using a Taqman® Real-time PCR system (Applied Biosystems Japan, Tokyo, Japan) with mouse RANK (Assay ID: Mm00437135_m1), human RANK (Assay ID: Hs00187192_m1), mouse NFATc1 (Assay ID: Mm00479445_m1), mouse integrin β 3 (Assay ID: Mm00443980_m1), mouse GAPDH (Assay ID: Mm03302249_g1), and human GAPDH (Assay ID: Hs02758991_g1) probes, which were provided by the manufacturer.

Techniques: Expressing, Transduction, Plasmid Preparation, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors

doi: 10.1371/journal.pone.0048795

Figure Lengend Snippet: A . BMMs under the adherent condition were harvested (0 h) and subsequently cultured again under adherent and non-adherent conditions in the presence or absence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) for the indicated periods. Relative expression levels of RANK mRNA were determined by real time RT-PCR. Data represent mean values of three independent experiments, with error bars indicating ± SD. ** P < 0.01 for adherent condition vs. non-adherent condition at the same time point. B . Left panel : BMMs grown under the adherent condition were transferred to the non-adherent condition and further cultured for the indicated time periods. Right panel : BMMs under the non-adherent condition were transferred to the adherent condition and further cultured for the indicated time periods. RANK mRNA expression was analyzed by RT-PCR. C . Left panel : comparison of RANK protein expression levels between BMMs cultured under the adherent condition and those transferred to the non-adherent condition. Right panel : Comparison of RANK protein levels between BMMs cultured under the non-adherent condition and those transferred to the adherent condition. RANK protein expression on the surface of BMMs was analyzed by flowcytometry.

Article Snippet: RANK, NFATc1, and integrin β 3 mRNA expressions were quantified using a Taqman® Real-time PCR system (Applied Biosystems Japan, Tokyo, Japan) with mouse RANK (Assay ID: Mm00437135_m1), human RANK (Assay ID: Hs00187192_m1), mouse NFATc1 (Assay ID: Mm00479445_m1), mouse integrin β 3 (Assay ID: Mm00443980_m1), mouse GAPDH (Assay ID: Mm03302249_g1), and human GAPDH (Assay ID: Hs02758991_g1) probes, which were provided by the manufacturer.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: PLoS ONE

Article Title: Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors

doi: 10.1371/journal.pone.0048795

Figure Lengend Snippet: A . BMMs were cultured in the presence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) with or without echistatin (10 -8 M). After culturing for 96 hours, cells were fixed and stained for TRAP ( A ), then TRAP activities were measured ( B ) and RANK expression levels evaluated ( C ). Data represent mean values of three independent experiments, with error bars indicating + SD. ** P <0.01.

Article Snippet: RANK, NFATc1, and integrin β 3 mRNA expressions were quantified using a Taqman® Real-time PCR system (Applied Biosystems Japan, Tokyo, Japan) with mouse RANK (Assay ID: Mm00437135_m1), human RANK (Assay ID: Hs00187192_m1), mouse NFATc1 (Assay ID: Mm00479445_m1), mouse integrin β 3 (Assay ID: Mm00443980_m1), mouse GAPDH (Assay ID: Mm03302249_g1), and human GAPDH (Assay ID: Hs02758991_g1) probes, which were provided by the manufacturer.

Techniques: Cell Culture, Staining, Expressing