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: 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: Bioengineering & Translational Medicine

Article Title: A high‐throughput biomimetic bone‐on‐a‐chip platform with artificial intelligence‐assisted image analysis for osteoporosis drug testing

doi: 10.1002/btm2.10313

Figure Lengend Snippet: Effect of OB‐dECM on osteogenic differentiation and maturation of IDG‐SW3 cells in a bone‐on‐a‐chip. (a) A schematic showing the strategy for cell–ECM interaction using Col/OB‐dECM simulating an in vivo osteon. (b and c) Relative gene expression levels of early osteocyte differentiation markers ( ALP , PDPN , and PHEX ) and late osteocyte differentiation markers ( DMP1 , SOST , and FGF23 ) in IDG‐SW3 cells according to the gel type (collagen or Col/OB‐dECM). Assays were performed on Days 7 and 14 after differentiation of osteocytes within gel ( N = 3). GAPDH was used as an internal control. (d) Morphological changes and alignment of IDG‐SW3 cells differentiated in collagen or Col/OB‐dECM hydrogels for 14 days ( N = 3). The area enclosed in a white box in the images above was enlarged in the images below. The cells were immunostained against F‐actin (Alexa Fluor 594, red). (e) Nuclear shape index (NSI) and alignment angle of IDG‐SW3 cells analyzed using the representative confocal images (Figure ) (Col, n = 66; Col/OB‐dECM, n = 70). (f) A schematic representing the implicit role of Cx43 on cellular function of osteocytes through cell–cell and cell–ECM interactions. (g) Cx43 protein level expressed in IDG‐SW3 cells according to the gel type. Assays were performed using ELISA on Days 7 and 14 after differentiation ( N = 3). All values are expressed as mean ± SD (* p < 0.05, ** p < 0.01). OB‐dECM, osteoblast‐derived decellularized extracellular matrix; ELISA, enzyme‐linked immunosorbent assay

Article Snippet: The protein levels of SOST were measured using a mouse SOST ELISA Kit (ALPCO).

Techniques: In Vivo, Gene Expression, Control, Cell Function Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Bioengineering & Translational Medicine

Article Title: A high‐throughput biomimetic bone‐on‐a‐chip platform with artificial intelligence‐assisted image analysis for osteoporosis drug testing

doi: 10.1002/btm2.10313

Figure Lengend Snippet: Effect of osteocyte and osteoblast coculture in a bone‐on‐a‐chip. (a–c) The osteocyte differentiation of IDG‐SW3 cells was affected upon coculture with MC3T3‐E1 cells. Relative gene expression levels of early differentiation markers ( ALP , PDPN , and PHEX ) and late differentiation markers ( DMP1 , SOST , and FGF23 ) in IDG‐SW3 cells monocultured or cocultured with MC3T3‐E1 cells on (a) Day 7 and (b) Day 14 after culture ( N = 3). GAPDH was used as an internal control. (c) The ALP protein level of IDG‐SW3 cells in mono or cocultured groups measured using an ALP quantification assay on Day 7 and Day 14 after culture ( N = 3). (d–g) The proliferation and osteoblastogenic differentiation of MC3T3‐E1 cells was affected upon coculture with IDG‐SW3 cells. (d) Representative immunofluorescence images showing staining against F‐actin (green) and nucleus (blue) of MC3T3‐E1 cells in monoculture or coculture groups on Day 7. (e) Proliferation rate of MC3T3‐E1 cells cocultured with IDG‐SW3 cells during 7 days measured using a CCK‐8 assay ( N = 3). (f and g) Relative gene expression levels of proliferation markers ( Cyclin D1 , c‐Myc , and CTNNB1 ) and osteoblast differentiation markers ( Runx2 , Osx , and OPG ) in MC3T3‐E1 cells monocultured or cocultured with IDG‐SW3 cells on (f) Day 7 and (g) Day 14 after culture ( N = 3). GAPDH was used as an internal control. All values are expressed as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). CCK‐8, Cell Counting Kit‐8

Article Snippet: The protein levels of SOST were measured using a mouse SOST ELISA Kit (ALPCO).

Techniques: Gene Expression, Control, Immunofluorescence, Staining, CCK-8 Assay, Cell Counting

Journal: Bioengineering & Translational Medicine

Article Title: A high‐throughput biomimetic bone‐on‐a‐chip platform with artificial intelligence‐assisted image analysis for osteoporosis drug testing

doi: 10.1002/btm2.10313

Figure Lengend Snippet: A comparison of the contribution of a bone‐on‐a‐chip and a Transwell system to bone maturation in osteocytes and osteoblasts. (a) Schematic illustration showing the difference in spatial structure between a bone‐on‐a‐chip and a Transwell system for coculturing osteocytes and osteoblasts. (b) Relative gene expression levels of early osteocyte markers ( ALP , PDPN , and PHEX ) and late osteocyte markers ( DMP1 , SOST , and FGF23 ) in IDG‐SW3 cells in each system on Day 7 ( N = 3). IDG‐SW3 cells embedded in gel (1 × 10 4 cells in 10 μl gel) were cocultured with osteoblasts (500 cells/well). All conditions were the same in both systems. GAPDH was used as an internal control. (c) Proliferation rate of MC3T3‐E1 cells cocultured with IDG‐SW3 cells during 7 days measured using a CCK‐8 assay ( N = 3). All values are expressed as mean ± SD (* p < 0.05, ** p < 0.01, **** p < 0.0001). CCK‐8, Cell Counting Kit‐8

Article Snippet: The protein levels of SOST were measured using a mouse SOST ELISA Kit (ALPCO).

Techniques: Comparison, Gene Expression, Control, CCK-8 Assay, Cell Counting

Journal: Bioengineering & Translational Medicine

Article Title: A high‐throughput biomimetic bone‐on‐a‐chip platform with artificial intelligence‐assisted image analysis for osteoporosis drug testing

doi: 10.1002/btm2.10313

Figure Lengend Snippet: Osteoporosis drug testing using bone‐on‐a‐chip. (a) A schematic showing the Wnt pathway related to bone formation, one of the target mechanisms for osteoporosis treatment. SOST, secreted by osteocytes, downregulates osteoblast proliferation (left image) whereas an anti‐SOST antibody (used as an osteoporosis drug) upregulates osteoblast proliferation through nuclear translocation of β‐catenin via the Wnt pathway (right image). (b) The overall sequential process for osteoporosis drug treatment in the bone‐on‐a‐chip. After 10 days of IDG‐SW3 maturation for SOST secretion, MC3T3‐E1 cells were cocultured for additional 4 days. Cells were treated twice with an osteoporosis drug on Days 10 and 12. (c) Representative images of MC3T3‐E1 cells in osteoporosis drug‐treated and untreated groups that were immunostained against β‐catenin (green) and nucleus (blue). (d) Relative average fluorescence intensity of β‐catenin in cells in each drug‐treated group compared to that in the control ( N = 20). (e) Relative β‐catenin nuclear translocation rate in drug‐treated group compared to that in the control ( N = 20). (f) The proposed CNN‐based deep learning architecture to perform osteoporosis drug testing. (g and h) Accuracy (left x ‐axis) and loss curves (right x ‐axis) of CNN training and validation for the (g) BN and (h) BNM datasets with successive epochs. (i) ROC curve analysis comparing classification results in BN and BNM datasets. AUC, area under the ROC curve; BN, β‐catenin and nucleus fluorescence image data; BNM, β‐catenin, nucleus, and their merged image data; CNN, convolutional neural network; ROC, receiver operating characteristic

Article Snippet: The protein levels of SOST were measured using a mouse SOST ELISA Kit (ALPCO).

Techniques: Translocation Assay, Fluorescence, Control, Biomarker Discovery

Journal: Bone Research

Article Title: Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

doi: 10.1038/boneres.2017.16

Figure Lengend Snippet: Deletion of Bmpr1a reduces SOST expression in osteocytes. ( a ) Representative images from immunohistochemistry of SOST on sections of the femur from littermate mice at P33. ( b ) Western blots with protein extracts from femurs and tibiae at P33. Each lane represents sample from a separate mouse. β-actin used as loading control. BM, bone marrow; CKO, Dmp1-Cre ; Bmpr1a f/f ; M.W., molecular weight markers; SOST, sclerostin.

Article Snippet: The sections were first blocked with 5% normal serum and then incubated in biotinylated sclerostin antibody (BAF1589, R&D Systems, Minneapolis, MN, USA) at 1:500 in blocking solution.

Techniques: Expressing, Immunohistochemistry, Western Blot, Control, Molecular Weight

Journal: Bone Research

Article Title: Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

doi: 10.1038/boneres.2017.16

Figure Lengend Snippet: Forced expression of SOST does not modify bone diameters in Bmpr1a-deficient mice. ( a ) Mating scheme. ( b ) X-ray radiography of the hindlimb from littermate mice at P33. Arrows denote restricted region in the proximal femur specific to CKO and SOST;CKO mice. Lines indicate expanded cancellous bone region. ( c and d ) μCT images ( c ) or quantification ( d ) of cortical bone acquired at the regions marked by the arrows or the equivalent regions in ( b ). * P <0.001, one-way ANOVA, n =5 for CTRL and CKO, n =6 for SOST, n =7 for SOST;CKO. f, femur; fi, fibula; SOST, sclerostin; t, tibia; μCT, micro-computed tomography.

Article Snippet: The sections were first blocked with 5% normal serum and then incubated in biotinylated sclerostin antibody (BAF1589, R&D Systems, Minneapolis, MN, USA) at 1:500 in blocking solution.

Techniques: Expressing, Micro-CT

Journal: Bone Research

Article Title: Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

doi: 10.1038/boneres.2017.16

Figure Lengend Snippet: Forced expression of SOST reduces cancellous bone mass in Bmpr1a-deficient mice. ( a ) μCT 3D reconstruction images of the metaphyseal region of distal femur in littermate mice at P33. Asterisk denotes marrow space. ( b ) μCT quantification of cancellous bone in the distal metaphyseal region of the femur. * P <0.05, Student’s t -test. ( c ) μCT quantification of total metaphyseal bone (including both cancellous and cortical bone). Note that the parameters may not accurately reflect cancellous bone properties especially in CTRL and SOST mice due to the inclusion of both cancellous and cortical bone in the analysis. * P <0.001, one-way ANOVA, n =5 for CTRL and CKO, n =6 for SOST, n =7 for SOST;CKO. ANOVA, analysis of variance; CTRL, control; SOST, sclerostin; 3D, three dimensional; μCT, micro-computed tomography.

Article Snippet: The sections were first blocked with 5% normal serum and then incubated in biotinylated sclerostin antibody (BAF1589, R&D Systems, Minneapolis, MN, USA) at 1:500 in blocking solution.

Techniques: Expressing, Control, Micro-CT

Journal: Bone Research

Article Title: Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

doi: 10.1038/boneres.2017.16

Figure Lengend Snippet: SOST expression partially rescues cancellous but not cortical bone phenotype caused by Bmpr1a deletion. ( a ) Serum CTX-I assays, n =3. ( b ) Representative images of H&E stained sections of the distal femur at P33. Boxed regions are shown at a higher magnification in lower panels. Arrow denotes accumulation of osteoblasts. ( c and d ) H&E staining ( c ) and quantification of osteocyte density ( d ) in cortical bone of the femur at P33. * P <0.001, two-way ANOVA, n =3. ( e ) Representative images of calcein double labeling at periosteal surface in P33 littermate mice. ( f ) Quantification of MAR. * P <0.001, owo-way ANOVA, n =3. ANOVA, analysis of variance; B, bone; M, marrow; SOST, sclerostin.

Article Snippet: The sections were first blocked with 5% normal serum and then incubated in biotinylated sclerostin antibody (BAF1589, R&D Systems, Minneapolis, MN, USA) at 1:500 in blocking solution.

Techniques: Expressing, Staining, Labeling

Journal: Bone Research

Article Title: Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

doi: 10.1038/boneres.2017.16

Figure Lengend Snippet: SOST expression is detected by immunofluorescence in both cancellous and cortical bone in SOST; CKO mice. Immunostaining was performed with non-immune IgG ( a and e ) or an antibody recognizing both murine and human sclerostin ( b – d and f – h ) on frozen sections of the femur. Blue, nuclear staining by DAPI; BM, bone marrow; double-headed arrow, cortical bone; GF, growth plate; red, antibody staining against both murine and human sclerostin; SOST, sclerostin.

Article Snippet: The sections were first blocked with 5% normal serum and then incubated in biotinylated sclerostin antibody (BAF1589, R&D Systems, Minneapolis, MN, USA) at 1:500 in blocking solution.

Techniques: Expressing, Immunofluorescence, Immunostaining, Staining

Journal: Bone Research

Article Title: Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

doi: 10.1038/boneres.2017.16

Figure Lengend Snippet: Forced expression of SOST partially corrects hyperproliferation caused by Bmpr1a deletion. ( a ) Representative images of distal femur labeled with EdU at P33. EdU signal is in green and DAPI nuclei staining in blue. ( b ) EdU labeling index over total cells in chondro-osseous junction. ( c ) Representative images of Osx immunofluorescence staining and EdU labeling at P33. Boxed regions in chondro-osseous junction below growth plate are shown at a higher magnification in lower panels. EdU is in green and Osx in red; Arrows denote double positive cells. ( d ) EdU labeling index among Osx+ preosteoblasts in chondro-osseous junction. ( e ) Negative control for Osx immunofluorescence staining. A non-immuno IgG detected no red signal on section from CTRL mouse. Region between dotted lines denotes chondro-osseous junction chosen for quantification [100 μm region immediately under growth plate (GP)]. GP: growth plate; M: marrow. * P <0.001, two-way ANOVA, n =3. ANOVA, analysis of variance; CTRL, control; SOST, sclerostin.

Article Snippet: The sections were first blocked with 5% normal serum and then incubated in biotinylated sclerostin antibody (BAF1589, R&D Systems, Minneapolis, MN, USA) at 1:500 in blocking solution.

Techniques: Expressing, Labeling, Staining, Immunofluorescence, Negative Control, Control

Journal: Bone Research

Article Title: Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

doi: 10.1038/boneres.2017.16

Figure Lengend Snippet: High bone mass Lrp5 mutant allele does not restore periosteal bone growth in Bmpr1a-deficient mice. ( a ) Mating scheme. ( b ) Representative X-ray images of hindlimbs of littermate mice at P33. f: femur; fi: fibula; t: tibia. Arrows denote smaller diameter in the proximal femur specific to CKO and HBM;CKO mice. Discs mark region of tibia (tibia-fibula junction) analyzed by μCT in ( c ). ( c ) Quantification of cortical bone parameters by μCT at region of tibia marked by disc in B. * P <0.01, two-way ANOVA, n =4. ( d ) Representative images for H&E staining of longitudinal sections through the proximal tibia. Note more bone in trabecular region (TB) and secondary ossification center (2°) in HBM than CTRL. ( e ) Quantification of trabecular bone parameters by μCT. ( f ) Model for Bmp signaling in osteoblast lineage cells. Bmp signaling via Bmpr1a directly promotes osteoblast activity in both trabecular and periosteal bone. Bmp also acts on osteocytes to induce SOST that in turn suppresses preosteoblast proliferation in trabecular bone region. On the other hand, production of periosteal osteoblasts is not altered by increased Wnt signaling. Trabecular versus periosteal osteoblast lineage is depicted in green versus blue. Red arrow and blocked arrow indicate stimulation and inhibition, respectively. Dashed line indicates potential action. ANOVA, analysis of variance; CTRL, control; OB, osteoblast; OC, osteocyte; PreOB, preosteoblast; 2°, secondary ossification center; μCT, micro-computed tomography.

Article Snippet: The sections were first blocked with 5% normal serum and then incubated in biotinylated sclerostin antibody (BAF1589, R&D Systems, Minneapolis, MN, USA) at 1:500 in blocking solution.

Techniques: Mutagenesis, Staining, Activity Assay, Inhibition, Control, Micro-CT

Journal: Journal of cellular biochemistry

Article Title: Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

doi: 10.1002/jcb.25432

Figure Lengend Snippet: Fig. 1. Sclerostin enhances adipocyte differentiation in 3T3-L1 cells. Confluent 3T3-L1 cells were incubated in differentiation media and treated with indicated doses of sclerostin at day 0 postinitiation of differentiation. Cells were fixed and lipid accumulation was monitored by oil red O staining at day 5 of differentiation (A). Lipid staining was extracted using isopropyl alcohol and oil red O accumulation quantified by measuring absorbance at 520 nm. Fold-increase in absorbance over nontreated cells is presented (B). After total RNA was extracted from the cells at day 5 of differentiation, adiponectin or peroxisome proliferator-activated receptor (PPAR) g mRNA level was determined by qRT- PCR (C). The lipoprotein lipase (LPL) and the fatty acid-binding protein 4 (Fabp4) mRNA levels were determined by qRT-PCR (D). b-actin was used as an endogenous control. Data are presented as means S.D.; n ¼ 3, , P < 0.05 versus absence of sclerostin (0).

Article Snippet: Recombinant mouse sclerostin and Wnt3a were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Staining, Quantitative RT-PCR, Binding Assay, Control

Journal: Journal of cellular biochemistry

Article Title: Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

doi: 10.1002/jcb.25432

Figure Lengend Snippet: Fig. 2. Sclerostin attenuates Wnt3a-inhibited adipocyte differentiation and expression of LRP5 and LRP6 in 3T3-L1 cells. (A and B) Confluent 3T3-L1 cells were incubated in differentiation media (DM þ) or none (DM ) and treated with indicated doses of sclerostin (20 ng/mL), Wnt3a (10 ng/mL), sclerostin (50 ng/mL) and Wnt3a (10 ng/mL), or vehicle (). At day 5, total RNA was extracted from the cells and adiponectin and PPARg mRNA levels were determined by qRT-PCR. GAPDH was used as an endogenous control. Data are presented as means S.D.; n ¼ 3, , P < 0.05. (B) Photograph of oil red O staining (left panel) and quantification of oil red O staining at day 5 (right panel). (C) LRP4, LRP5, and LRP6 mRNA expression were determined by RT-PCR in 3T3-L1 preadipocytes (A) or MC3T3-E1 osteoblasts (O). b-actin was used as a positive control. Lane M represents the size marker (100-bp ladder).

Article Snippet: Recombinant mouse sclerostin and Wnt3a were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Control, Staining, Reverse Transcription Polymerase Chain Reaction, Positive Control, Marker

Journal: Journal of cellular biochemistry

Article Title: Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

doi: 10.1002/jcb.25432

Figure Lengend Snippet: Fig. 3. Sclerostin regulates C/EBPb expression but not cell proliferation and or caspase-3/7 activity in 3T3-L1 cells. (A) Confluent 3T3-L1 cells were incubated in differentiation media and treated with sclerostin (20 ng/mL). After 24 h, the level of C/EBPb protein in the cells was determined by Western blot analysis. (B) Confluent 3T3-L1 cells were incubated in differentiation media and treated with sclerostin (20 ng/mL), and then cultured for further 24 h. DNA synthesis of 3T3-L1 cells was measured by BrdU incorporation using an ELISA kit. BrdU incorporation in the absence of sclerostin is adjusted to 1. (C) Confluent 3T3-L1 cells were incubated in differentiation media and treated with sclerostin (20 ng/mL). After 4 h, cellular caspase-3/7 activities were measured. Fold-increase in activity was calculated based on activity measured in control (absence) cells. Each assay represents a separate experiment performed in triplicate. Data are presented as means S.D; n ¼ 3; n.s. (no significant difference).

Article Snippet: Recombinant mouse sclerostin and Wnt3a were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Activity Assay, Incubation, Western Blot, Cell Culture, DNA Synthesis, BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of cellular biochemistry

Article Title: Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

doi: 10.1002/jcb.25432

Figure Lengend Snippet: Fig. 4. Effects of TAZ knockdown on sclerostin-mediated adipocyte differentiation in 3T3-L1 cells. (A) 3T3-L1 cells were incubated in differentiation media and then transiently cotransfected in 24-well plates with a TAZ reporter plasmid 8xGTIIC-Lux. Then cells were treated with sclerostin (20 ng/mL), Wnt3a (10 ng/mL), sclerostin (20 ng/mL), and Wnt3a (10 ng/mL) or vehicle () for 6 h, after which luciferase activity was determined. Normalized luciferase activity is shown as the ratio of luciferase activity relative to 8xGTIIC-Lux with vehicle, which is set to a value of 1. (B) Confluent 3T3-L1 cells were incubated in differentiation media and treated with sclerostin (20 ng/mL), Wnt3a (10 ng/ mL), sclerostin (50 ng/mL) and Wnt3a (10 ng/mL) or vehicle () for 5 days. Total RNA was extracted from the cells and then ctgf mRNA level was determined by qRT-PCR. (C, D, E, and F) 3T3-L1 cells were transiently transfected with TAZ siRNA (siTAZ) or control siRNA (siCont) (both at 10 nM) at day 0. Then cells were treated with sclerostin (20 ng/mL) or vehicle (). At day 2, the levels of TAZ protein in the cells were determined by Western blot analysis. (D) Photographs of oil red O staining and (E) quantification of oil red O staining in 3T3-L1 cells at day 6. Graph showing fold-increase in absorbance over nontreated cells. (F) A qRT-PCR was performed to quantify mRNA expression level of adiponectin. b-actin was used as an endogenous control. Data are presented as means S.D; n ¼ 3; , P < 0.05.

Article Snippet: Recombinant mouse sclerostin and Wnt3a were purchased from R&D Systems (Minneapolis, MN).

Techniques: Knockdown, Incubation, Plasmid Preparation, Luciferase, Activity Assay, Quantitative RT-PCR, Transfection, Control, Western Blot, Staining, Expressing

Journal: Journal of cellular biochemistry

Article Title: Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

doi: 10.1002/jcb.25432

Figure Lengend Snippet: Fig. 5. Effects of TAZ over-expression on sclerostin-mediated adipocyte differentiation in 3T3-L1 cells. 3T3-L1 cells were transiently transfected in 24-well plates with a TAZ expression plasmid pTAZ or empty vector pcDNA3 (both at 0.1 mg/well). After one day, cells were treated with sclerostin (20 ng/mL) or vehicle () (day 0). The levels of TAZ protein in the cells were determined by Western blot analysis at day 2 (A). Photographs of oil red O staining (B) and quantification of oil red O staining (C) in 3T3-L1 cells at day 6. Total RNA was extracted from the cells at day 6 and then a qRT-PCR was performed to quantify mRNA expression level of adiponectin (D). b-actin was used as an endogenous control. Data are presented as means S.D; n ¼ 3; n.s. (no significant difference).

Article Snippet: Recombinant mouse sclerostin and Wnt3a were purchased from R&D Systems (Minneapolis, MN).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: PPARG in osteocytes is essential for sclerostin expression, bone mass, marrow adiposity and TZD-induced bone loss

doi: 10.1101/2020.09.13.295378

Figure Lengend Snippet: Correlation between PPARG and sclerostin levels in osteocytes. A. and B. Levels of PPARG KO and sclerostin proteins in OT isolated from 6.5 mo old male γOT KO and Ctrl mice and either analyzed KO on separate (A) or the same (B) Western blot membrane. (γOT KO n=5; Ctrl n=4). C) Pearson correlation analysis of PPARG and sclerostin protein levels shown in panel B. Coefficient of determination (R 2 =0.9821) was calculated based on bands density assessed by Image J and normalized to b-actin. D. KO Sclerostin levels measured by ELISA in sera of γOT KO (n=4) and Ctrl (n=7) 6 mo old male mice. Statistical significance was calculated using parametric unpaired Student’s t test.

Article Snippet: Sclerostin levels after depletion were quantified using SOST ELISA kit (Quantikine ELISA, R&D Systems, Cat#MSST00).

Techniques: Isolation, Western Blot, Membrane, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: PPARG in osteocytes is essential for sclerostin expression, bone mass, marrow adiposity and TZD-induced bone loss

doi: 10.1101/2020.09.13.295378

Figure Lengend Snippet: PPARG positively regulates sclerostin expression. A. Schematic positioning of JASPAR projected PPARG response elements (PPREs) upstream of sclerostin transcription start site (TSS). B. Chromatin immunoprecipitation (ChIP) assay end point agarose gel image (full image of the gel is presented in Suppl. Fig. 1). ChIP was performed on osteocyte-like MLO-Y4 cells targeting PPRE3 and PPRE14/15. Cells were treated for 24h with either vehicle (V), or 1µM rosiglitazone (R) or combination of 1µM R and 10µM GW9662 antagonist (R+GW) and ChIP assays were performed, as described in Material and Methods. S – sonicated lysate (no antibody pulldown). C. Expression of Sost mRNA in MLO-Y4 cells treated for 3 days with either vehicle (V), or rosiglitazone (R), or combination of rosiglitazone and GW9662 (R+GW) at the same doses as in (B). One-way ANOVA followed by Tukey’s post hoc analysis was performed for significance calculation. D. Western blot of sclerostin protein levels in MLO-Y4 cells treated as in (C). Sclerostin protein levels were normalized to β-actin levels measured as band density using Image J and relative levels of expression had been calculated and shown below Western blot images.

Article Snippet: Sclerostin levels after depletion were quantified using SOST ELISA kit (Quantikine ELISA, R&D Systems, Cat#MSST00).

Techniques: Expressing, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Sonication, Western Blot

Journal: bioRxiv

Article Title: PPARG in osteocytes is essential for sclerostin expression, bone mass, marrow adiposity and TZD-induced bone loss

doi: 10.1101/2020.09.13.295378

Figure Lengend Snippet: Osteocyte derived sclerostin positively contributes to marrow adipocyte differentiation. A. Schematic showing experimental design of co-culture of BMSC with intact or sclerostin depleted conditioned medium (CM). One group of adherent BMSC culture received IgG depleted CM from primary osteocytes (control group) while the other group received sclerostin depleted CM (group of interest). B. ELISA measurements of sclerostin level in CM after anti-SOST antibody mediated depletion. C. Expression of adipocytic and osteoblastic gene markers in adherent BMSCs treated with CM from primary osteocytes IgG or sclerostin (Ab-Scl) depleted. Statistical significance was calculated using parametric unpaired Student’s t test.

Article Snippet: Sclerostin levels after depletion were quantified using SOST ELISA kit (Quantikine ELISA, R&D Systems, Cat#MSST00).

Techniques: Derivative Assay, Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay, Expressing