Journal: Osteoarthritis and cartilage

Article Title: Sex Differences in the Estrogen-Dependent Regulation of Temporomandibular Joint Remodeling in Altered Loading

doi: 10.1016/j.joca.2016.11.008

Figure Lengend Snippet: The data represent WT and ERβKO mice under normal load or decreased occlusal loading with either placebo or estradiol treatment. Specifically, the labels indicate the following: NL = normal load and placebo; DOL = decreased occlusal loading and placebo; Esd = normal load and estradiol; DOL + Esd = decreased occlusal loading and estradiol. Representative Col2 immunohistochemical images (A), representative safranin O images (B) and gene expression of Col2 (C), Sox9 (D), Pthrp (E) and Ihh (F) and Sost (G) are shown. For gene expression, n=6 mice were utilized for all groups and mandibular condylar cartilage from left and right were pooled together. Statistical significance was determined by a two-way ANOVA followed by posthoc analysis with the Bonferonni method with p < 0.05. Exact p values are listed above the bars that denote significance.

Article Snippet: Gene expression was analyzed for the following chondrocyte markers: parathyroid hormone-related peptide ( PTHrP – Mm00436057_m1), SRY-box containing gene 9 ( Sox9 – MM00448840_m1), collagen type II ( Col 2a1 – Mm00491889_m1), collagen type X ( Col 10a1 – Mm00487041_m1), sclerostin ( Sost – Mm00470479_m1), indian hedgehog ( Ihh – Mm00439613_m1), estrogen receptor α ( Esr1 – Mm00433149_m1), and estrogen receptor β ( Esr2 – Mm00599819) .

Techniques: Immunohistochemical staining, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity

doi: 10.1074/jbc.m301716200

Figure Lengend Snippet: FIG. 1. Expression of sclerostin during mouse development. A, frontal section of E11 head. The only tissue expressing sclerostin mRNA is the endothelium of the pharyngeal artery (arrow). B, intense punc- tuated expression is seen at the sites of mandibular and maxillary bones at E15 (arrows). Meckel’s cartilage is negative. C, Bsp mRNA expression in osteoblasts marks the extent of bone formation at E16. D, scattered sclerostin mRNA-expressing cells are present on the surfaces of all bones in the head of a newborn mouse. The cells are abundant in the bone surrounding the growing tooth germs (arrows). E, in develop- ing long bones of E16 and E18 mouse embryos, sclerostin mRNA ex- pression is seen in the perichondrium and periosteum as well as in trabecular bone but not in the cartilage. White grains in dark field images indicate the expression of sclerostin mRNA. F, in a section through the ribs of E17 embryo, sclerostin mRNA is expressed in iso- lated large cells in the cartilage perichondrium. G, in the liver of E12 embryo, sclerostin mRNA expression is intense in hematopoietic cells. M, molar tooth germ; T, tongue; MC, Meckel’s cartilage; R, resting chondrocytes; P, proliferating chondrocytes; H, hypertrophic chondro- cytes; TB, trabecular bone; PC, perichondrium; PO, periosteum; RC, rib cartilage.

Article Snippet: After the cells had reached confluence, the medium was replaced with -minimum essential medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 24 h. The cells were then cultured in -minimum essential medium containing 1% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, 10 ng/ml recombinant human BMP2 (25 ng/ml), BMP4 (10 ng/ml), BMP6 (10 ng/ml), or BMP7 (25 ng/ml) protein (R&D Systems), either and 0–100 ng/ml recombinant mouse sclerostin protein or 100 ng/ml recombinant mouse noggin/Fc chimera (R&D Systems) for 72 h. Cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl-containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2.

Techniques: Expressing

Journal: Journal of Biological Chemistry

Article Title: Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity

doi: 10.1074/jbc.m301716200

Figure Lengend Snippet: FIG. 2. Codistribution of sclerostin and MMP-9. The patterns of cells expressing sclerostin mRNA and MMP-9 (a marker of osteoclasts) mRNA are similar in the mandibular bone at E15 (A and B), in calvarial bone in the newborn mouse (NB) (C and D), and in the mandibular bone around the tooth germ in the newborn mouse (E and F).

Article Snippet: After the cells had reached confluence, the medium was replaced with -minimum essential medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 24 h. The cells were then cultured in -minimum essential medium containing 1% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, 10 ng/ml recombinant human BMP2 (25 ng/ml), BMP4 (10 ng/ml), BMP6 (10 ng/ml), or BMP7 (25 ng/ml) protein (R&D Systems), either and 0–100 ng/ml recombinant mouse sclerostin protein or 100 ng/ml recombinant mouse noggin/Fc chimera (R&D Systems) for 72 h. Cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl-containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2.

Techniques: Expressing, Marker

Journal: Journal of Biological Chemistry

Article Title: Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity

doi: 10.1074/jbc.m301716200

Figure Lengend Snippet: FIG. 3. Detection of recombinant mouse sclerostin protein. A, the cell lysate and culture medium of cells expressing recombinant mouse sclerostin protein were separated by SDS-polyacrylamide gel electrophoresis under reducing (with 1,4-dithiothreitol; DTT) or non- reducing (without 1,4-dithiothreitol; DTT) conditions followed by Western blotting analysis with anti-E tag antibodies. B, purified recom- binant mouse sclerostin (0.35 g) was separated by SDS-polyacryl- amide gel electrophoresis under reducing conditions and subjected to protein staining.

Article Snippet: After the cells had reached confluence, the medium was replaced with -minimum essential medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 24 h. The cells were then cultured in -minimum essential medium containing 1% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, 10 ng/ml recombinant human BMP2 (25 ng/ml), BMP4 (10 ng/ml), BMP6 (10 ng/ml), or BMP7 (25 ng/ml) protein (R&D Systems), either and 0–100 ng/ml recombinant mouse sclerostin protein or 100 ng/ml recombinant mouse noggin/Fc chimera (R&D Systems) for 72 h. Cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl-containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2.

Techniques: Recombinant, Expressing, Polyacrylamide Gel Electrophoresis, Western Blot, Purification, Nucleic Acid Electrophoresis, Staining

Journal: Journal of Biological Chemistry

Article Title: Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity

doi: 10.1074/jbc.m301716200

Figure Lengend Snippet: FIG. 4. Effects of sclerostin and nog- gin on alkaline phosphatase activity in MC3T3-E1 cells induced by BMPs. MC3T3-E1 cells were treated with BMP6 (10 ng/ml) (A), BMP7 (25 ng/ml) (B), BMP2 (25 ng/ml) (C), or BMP4 (10 ng/ml) (D) and different concentrations of mouse recombinant sclerostin or 100 ng/ml nog- gin for 72 h. After treatment, alkaline phosphatase activity in MC3T3-E1 cells was determined. Results are the means S.D. for five independent wells.

Article Snippet: After the cells had reached confluence, the medium was replaced with -minimum essential medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 24 h. The cells were then cultured in -minimum essential medium containing 1% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, 10 ng/ml recombinant human BMP2 (25 ng/ml), BMP4 (10 ng/ml), BMP6 (10 ng/ml), or BMP7 (25 ng/ml) protein (R&D Systems), either and 0–100 ng/ml recombinant mouse sclerostin protein or 100 ng/ml recombinant mouse noggin/Fc chimera (R&D Systems) for 72 h. Cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl-containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2.

Techniques: Activity Assay, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity

doi: 10.1074/jbc.m301716200

Figure Lengend Snippet: FIG. 5. Binding of sclerostin to BMP6. Mouse recombinant sclerostin was fixed on the carboxylmethyl sensor tip. The binding of different concentrations of BMP6 on the tip was analyzed using the BIAcore 2000 system.

Article Snippet: After the cells had reached confluence, the medium was replaced with -minimum essential medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 24 h. The cells were then cultured in -minimum essential medium containing 1% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, 10 ng/ml recombinant human BMP2 (25 ng/ml), BMP4 (10 ng/ml), BMP6 (10 ng/ml), or BMP7 (25 ng/ml) protein (R&D Systems), either and 0–100 ng/ml recombinant mouse sclerostin protein or 100 ng/ml recombinant mouse noggin/Fc chimera (R&D Systems) for 72 h. Cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl-containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2.

Techniques: Binding Assay, Recombinant

Journal: Cells

Article Title: Sclerostin Alters Tumor Cell Characteristics of Oral Squamous Cell Carcinoma and May Be a Key Player in Local Bone Invasion

doi: 10.3390/cells13020137

Figure Lengend Snippet: Immunohistochemical staining protocol.

Article Snippet: Sclerostin , Mouse, monoclonal, clone AbD09097_h/mIgG 2a, 1:1200 , HIER (pH 9) , Dako EnVision FLEX , BioRad, Hercules, CA, USA (HCA230Z).

Techniques: Immunohistochemical staining, Staining

Journal: Clinical Journal of the American Society of Nephrology

Article Title: Sclerostin and Dickkopf-1 in Renal Osteodystrophy

doi: 10.2215/cjn.06550810

Figure Lengend Snippet: Figure 1. | (A and B) Scatter plot of serum concentrations of sclerostin (pg/ml) at different levels of Ac.f. (A) and different levels of BFR/BS (B).

Article Snippet: Fifty microliters of serum was loaded per well, incubated overnight at 4°C, washed, and incubated for 1 hour at 37°C, followed by incubation at 4°C for 1 hour with a biotinylated polyclonal anti-sclerostin antibody (BAF 1406; R&D Systems) diluted to a concentration of 0.5 g/ml in dilution buffer.

Techniques: