Journal: Frontiers in Immunology

Article Title: In silico design of novel precision vaccine targeting sclerostin epitopes for osteoporosis prevention and treatment

doi: 10.3389/fimmu.2025.1644437

Figure Lengend Snippet: Screening and analysis of high-affinity epitopes on SOST. (A) ELISA experiments were conducted to identify SOST fragments with strong binding affinity for ROMO, revealing that SOST 114–143 and SOST 143–173 exhibit significantly higher affinity ( P <0.01). (B) A schematic diagram delineating the binding functional regions associated with the high-affinity fragments of SOST. (C) ELISA results indicate that SOST 131–163 displays the highest affinity for ROMO ( P <0.01), thereby identifying it as a potent functional epitope of SOST. (D-a) SOST 131–163 fragment (highlighted in yellow) is located within the loop3 domain of SOST protein. (D-b) Docking studies indicate that SOST 131–163 fragment interacts with ROMO light chain, yielding a binding free energy of -25.8 kcal/mol and an interface area of 712.9 Ų. (D-c) Additionally, SOST 131–163 fragment can bind to the ROMO heavy chain, resulting in a binding free energy of -33.19 kcal/mol and an interface area of 451.6 Ų. (E) CTL epitopes within SOST 131–163 sequence include two strong binder epitopes and four weak binder epitopes. (F) HTL epitopes in SOST 131–163 sequence comprise one strong binder epitope and four weak binder epitopes. Predictions of B cell epitopes for SOST 131–163 sequence are illustrated, including predicted linear B cell epitopes (G) and predicted discontinuous B cell epitopes (H) .

Article Snippet: In brief, 1 μg/mL of human SOST protein (MedChemExpress Inc.) was coated onto the wells of MaxiSorp microtiter plates (Thermo Fisher Scientific Inc.) and incubated overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Functional Assay, Sequencing

Journal: Bone

Article Title: A Xenograft Model to Evaluate the Bone Forming Effects of Sclerostin Antibody in Human Bone Derived from Pediatric Osteogenesis Imperfecta Patients

doi: 10.1016/j.bone.2019.115118

Figure Lengend Snippet: Immunohistochemistry with fluorescence (IHC-F) following removal from the host at 2 and 4 weeks treated (SclAb) and untreated. Human osteogenesis imperfecta (OI) implants were dual IHC-F stained to probe for the presence of Osterix (Osx; green) and human mitochondria (hMito; red) (A-D) and on serial sections, Sclerostin (yellow) and hMito (red) (E-H). In all cases, hMito was used to indicate donor derived cells and Osx or sclerostin primary antibody (validated sensitivity to both mouse and human antigens) were used to probe all instances of expression (both host and donor). Zoomed insets (1) depict lining cells expressing Osx (A-D) and osteocytes expressing sclerostin (E-H). Representative Hematoxylin and Eosin stained bone acquired at baseline (I). Images were acquired at 40x (50 μm scale bar). DAPI= nuclear stain (blue). Panel represents data from one OI patient (OI 6, Type III/IV OI) who yielded cortical-derived bone samples (I), hematoxylin and eosin (H&E) stained implant acquired at 20x with a 250 μm scale bar.

Article Snippet: Sections were incubated with the primary anti-hMito antibody (MAB1273, EMD Millipore) at a 1:200 dilution and either a primary polyclonal rabbit anti-Osx antibody (ab22552, Abcam; 1:400) or primary polyclonal rabbit anti-sclerostin antibody (bs-10200r, Bioss; 1:200) overnight at 4°C.

Techniques: Immunohistochemistry, Fluorescence, Staining, Derivative Assay, Expressing

Journal: Diabetes/Metabolism Research and Reviews

Article Title: Fibre‐Enriched High‐Carbohydrate (FEHC) Diet Modulates Inflammation Without Affecting Bone Health in Older Women With Obesity: A Randomised Clinical Trial

doi: 10.1002/dmrr.70089

Figure Lengend Snippet: Gene expression analysis by Real‐time PCR of Wnt signaling and bone metabolism–related genes in bone tissue at T1 in a fiber‐enriched high‐carbohydrate diet (FEHC, green bars) and a control diet (CD, pink bars) group. (A) Wnt family member 5A (WNT5A). (B) Wnt family member 10B (WNT10B). (C) Sclerostin (SOST). (D) Dickkopf WNT signaling pathway inhibitor 1 (DKK1). (E) Lymphoid enhancer‐binding factor 1 (LEF1). (F) Osteocalcin (OCN). (G) Runt‐related transcription factor 2 (RUNX2). (H) Collagen type I alpha 1 chain (COL1A1). Data are presented as median values with interquartile ranges (IQR). Statistical analysis was performed using two‐way ANOVA followed by Tukey’s multiple comparisons test. Comparisons were considered statistically significant at p < 0.05.

Article Snippet: Gene expression of WNT5A (Hs00998537_m1), WNT10B (Hs00928823_m1), SOST (Hs00228830_m1), IGF‐1 (Hs01547656_m1), IL‐6 (Hs00174131_m1), IL‐8 (Hs00174103_m1), IL‐10 (Hs00961622_m1), TNFα (Hs00174128_m1), ADIPOQ (Hs00605917_m1), OCN (Hs01587814_g1), RUNX2 (Hs00231692_m1), DKK‐1 (Hs00183740_m1), LEF‐1 (Hs01547250_m1), COL1A1 (Hs00164004_m1) were evaluated in bone and muscle tissue biopsies.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Control, Binding Assay

Journal: Diabetes/Metabolism Research and Reviews

Article Title: Fibre‐Enriched High‐Carbohydrate (FEHC) Diet Modulates Inflammation Without Affecting Bone Health in Older Women With Obesity: A Randomised Clinical Trial

doi: 10.1002/dmrr.70089

Figure Lengend Snippet: Gene expression analysis by Real‐time PCR of Wnt signaling and bone metabolism–related genes in muscle tissue at T1 in a fiber‐enriched high‐carbohydrate diet (FEHC, green bars) and a control diet (CD, pink bars) group. (A) Wnt family member 5A (WNT5A). (B) Wnt family member 10B (WNT10B). (C) Sclerostin (SOST). (D) Dickkopf WNT signaling pathway inhibitor 1 (DKK1). (E) Lymphoid enhancer‐binding factor 1 (LEF1). (F) Osteocalcin (OCN). (G) Runt‐related transcription factor 2 (RUNX2). (H) Collagen type I alpha 1 chain (COL1A1). Data are presented as median values with interquartile ranges (IQR). Statistical analysis was performed using two‐way ANOVA followed by Tukey’s multiple comparisons test. Comparisons were considered statistically significant at p < 0.05.

Article Snippet: Gene expression of WNT5A (Hs00998537_m1), WNT10B (Hs00928823_m1), SOST (Hs00228830_m1), IGF‐1 (Hs01547656_m1), IL‐6 (Hs00174131_m1), IL‐8 (Hs00174103_m1), IL‐10 (Hs00961622_m1), TNFα (Hs00174128_m1), ADIPOQ (Hs00605917_m1), OCN (Hs01587814_g1), RUNX2 (Hs00231692_m1), DKK‐1 (Hs00183740_m1), LEF‐1 (Hs01547250_m1), COL1A1 (Hs00164004_m1) were evaluated in bone and muscle tissue biopsies.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Control, Binding Assay

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: 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