Journal: Cell reports

Article Title: Osteocyte intrinsic TGFβ signaling regulates bone quality through perilacunar/canalicular remodeling

doi: 10.1016/j.celrep.2017.10.115

Figure Lengend Snippet: (A–D) qPCR analysis of PLR genes Mmp13, Mmp14 and Ctsk and Serpine1 upon TGFβ (5ng/mL) treatment in MLO-Y4 (A, B) and OCY454 (C, D) cells. (n=3 replicates/group). (E, F) Intracellular pH (pHi) of MLO-Y4 cells after 3 days of TGFβ (5ng/ml), TβRI inhibitor SB-431542 (10 μM), or recombinant sclerostin (rhSCL, 10 ng/ml). The representative image (E) shows the shift in the emission peak from 580 nm to 640 nm after TGFβ treatment of MLO-Y4 cells. Scale bar, 100 μm). TGFβ-induced acidification is blocked by SB-431542 (F) (n=4 replicates/group). Error bars indicate mean ± SD of 3 independent experiments, *p<0.05 different from control mRNA, a-p<0.05 different from control pHi, b-p<0.05 different from TGFβ pHi, and c-p<0.05 different from rhSCL pHi. Statistics calculated from Student’s t test.

Article Snippet: For treatment, cells were cultured in α-MEM containing 0.5–1% fetal bovine serum, supplemented with 5 ng/ml TGFβ1 (Humanzyme, HZ-1011), 10 μM SB431542 (Sigma, S4317) or 10 ng/ml recombinant human sclerostin (rhSCL, R&D Systems) for the indicated times.

Techniques: Recombinant, Control

Journal: Cell reports

Article Title: Osteocyte intrinsic TGFβ signaling regulates bone quality through perilacunar/canalicular remodeling

doi: 10.1016/j.celrep.2017.10.115

Figure Lengend Snippet: (A, B) TβRII-stained osteocytes (A) (arrow, scale bar, 50 μm) in the femoral cortical bone from WT and TβRIIocy−/− mice (8-week old males) were quantified as percentage of positively stained osteocytes normalized to total bone area (B) (n=5 mice/group) (C) qPCR analysis of TβRII and Serpine1 in WT and TβRIIocy−/− femoral bones. (n=8–10 mice/group). (D, E) Silver nitrate stained images of WT and TβRIIocy−/− femoral cortical bone shows the osteocyte lacuno-canalicular network (D) and canalicular length (E) (scale bar, 20 μm, n=5 mice/group). (F, G) qPCR analysis of PLR genes, Mmp2, Mmp13, Mmp14, Ctsk, and Acp5 (F) and OCY-specific genes, Sost, Dmp1 and Phex (G) in the WT and TβRIIocy−/− bones (n=8–10 mice/group) (H, I) IHC of MMP13, MMP14, CTSK and H&E staining of WT and TβRIIocy−/− femoral cortical bone. Arrows in the image indicate positively stained osteocytes (H) that were quantified and normalized to total bone area (I), (n=4 mice/group).(J–M) SRμT shows volume (J), degree of anisotropy (K), orientation (L) and mineralization (N) of osteocyte lacunae of WT and TβRIIocy−/− bone (n=3–4 mice/group). Error bars indicate mean ± SEM with *p<0.05 compared to WT from Student’s t test.

Article Snippet: For treatment, cells were cultured in α-MEM containing 0.5–1% fetal bovine serum, supplemented with 5 ng/ml TGFβ1 (Humanzyme, HZ-1011), 10 μM SB431542 (Sigma, S4317) or 10 ng/ml recombinant human sclerostin (rhSCL, R&D Systems) for the indicated times.

Techniques: 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

Journal: JCI insight

Article Title: Sclerostin inhibition alleviates breast cancer-induced bone metastases and muscle weakness.

doi: 10.1172/jci.insight.125543

Figure Lengend Snippet: Figure 1. Breast cancer–derived sclerostin inhibits Wnt signaling in osteoblasts. (A and B) Calvarial cells were differentiated into osteoblasts in the presence of control medium or cancer-conditioned medium (CM) collected from MDA-MB-231 metastatic breast cancer cells. Osteoblast differen- tiation was determined by quantification of Runx2 and osteocalcin (Ocn) gene expression (A) and by alizarin red S staining (B) (n = 4 independent experiments). (C) Calvarial osteoblasts were cultured in the presence of the indicated amount of MDA-MB-231–derived CM. Wnt signaling activity was determined by TOPflash reporter assay (n = 4 independent experiments). (D) Sclerostin mRNA expression was quantified in nonmetastatic MCF-7 and metastatic MDA-MB-231 breast cancer cells by qRT-PCR (n = 6 independent experiments). (E) SOST mRNA expression was analyzed in breast cancer tissue from 48 patients. Proportion of sclerostin-positive and sclerostin-negative tissue samples is shown for all patients and for triple-negative (ER–, PR–, HER–) and in hormone receptor–negative (ER–, PR–, HER+) patients. All, n = 48; ER–, PR–, HER-, n = 9; ER–, HER–, HER+, n = 7. (F) Wnt signaling activity in calvarial osteoblasts cultured with control medium or with CM from MDA-MB-231 cells transfected with scrambled control siRNA (si-Ctrl CM) or siRNA against sclerostin (si-Sclerostin CM) (n = 6 independent experiments). (G) Wnt signaling activity in calvarial osteoblasts isolated from mice heterozygous for the Lrp5 mutation G171V (Lrp5-G171V+/T) and from control littermates (Lrp5-G171V+/+) stimulated with control medium or cancer CM (n = 4 independent experiments). Data are presented as mean ± SEM. Two-tailed Student’s t test was used to compare 2 groups (A and D), and ANOVA followed by Tukey’s post hoc analysis was used to compare 3 or more groups (C, F, and G); *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: MDA-MB-231 cells were transfected with scrambled control siRNA or siRNA against sclerostin (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Control, Gene Expression, Staining, Cell Culture, Activity Assay, Reporter Assay, Expressing, Quantitative RT-PCR, Transfection, Isolation, Mutagenesis, Two Tailed Test

Journal: JCI insight

Article Title: Sclerostin inhibition alleviates breast cancer-induced bone metastases and muscle weakness.

doi: 10.1172/jci.insight.125543

Figure Lengend Snippet: Figure 2. Pharmacological inhibition of sclerostin reduces bone metastatic burden in mice. MDA-MB-231 breast cancer cells stably expressing the lucifer- ase gene were injected into the left ventricle of 8-week-old female immune-compromised SCID mice. Micrometastases were detected 2 weeks after breast cancer cell injection by bioluminescence imaging (BLI). Mice were randomized and received either vehicle (n = 8) or anti-sclerostin antibody (Scl-Ab; n = 8) once a week for 4 weeks. (A and B) Tumor growth in bone was visualized after 4 weeks of treatment (A) and quantified (B) by BLI. Values are represented in a log10 scale. (C and D) Quantification of the metastasis area (C) in the tibia (D) of cancer-bearing mice treated with vehicle (n = 16 tibiae) or Scl-Ab (n = 16 tibiae) using histological sections. Scale bar: 1 mm. (E and F) Quantification of human leukocyte antigen (HLA) mRNA expression in the lung (E) and brain (F) by qRT-PCR (n = 8). (G) Kaplan-Meier survival curve of cancer-bearing mice treated with vehicle (n = 6) or Scl-Ab (n = 6). Data are presented as mean ± SEM. Two groups were compared using 2-tailed Student’s t test; *P < 0.05.

Article Snippet: MDA-MB-231 cells were transfected with scrambled control siRNA or siRNA against sclerostin (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Inhibition, Stable Transfection, Expressing, Injection, Imaging, Quantitative RT-PCR

Journal: JCI insight

Article Title: Sclerostin inhibition alleviates breast cancer-induced bone metastases and muscle weakness.

doi: 10.1172/jci.insight.125543

Figure Lengend Snippet: Figure 4. Inhibition of sclerostin prevents breast cancer–induced loss of muscle function. (A) Specific force of the extensor digitorum longus (EDL) muscle from healthy mice without treatment (n = 5), or treated with vehicle (n = 10) or anti-sclerostin antibody (Scl-Ab, n = 10); and from cancer-bearing mice treated with vehicle (n = 8) or Scl-Ab (n = 8). N, newtons. (B) Endurance of the EDL muscle of mice with bone metastases treated with vehicle (n = 8) or Scl-Ab (n = 8). max, maximum. (C) Succinate dehydroxygenase–stained (SDH-stained) tibialis anterior muscle sections from healthy mice without treatment or with vehicle or Scl-Ab treatment, as well as from mice with bone metastases treated with vehicle or Scl-Ab stained. Two representative muscles are shown per group. Scale bar: 50 μm. (D) Quantification of the cross-sectional area (CSA) of all muscle fibers, oxidative fibers, and nonoxida- tive fibers using SDH-stained muscle sections from healthy mice without treatment (n = 5), or with vehicle (n = 10) or Scl-Ab treatment (n = 10); and from cancer-bearing mice treated with vehicle (n = 8) or Scl-Ab (n = 8). Data are presented as mean ± SEM. Three or more groups were compared using ANOVA followed by Tukey’s post hoc analysis; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: MDA-MB-231 cells were transfected with scrambled control siRNA or siRNA against sclerostin (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Inhibition, Staining, Muscles

Journal: JCI insight

Article Title: Sclerostin inhibition alleviates breast cancer-induced bone metastases and muscle weakness.

doi: 10.1172/jci.insight.125543

Figure Lengend Snippet: Figure 5. Treatment with an anti-sclerostin antibody reverses breast cancer–induced activation of NF-κB signaling and increased number of Pax7-positive cells. (A) Immunoblot analysis of phosphorylated IKKα and IKKβ (p-IKKα and p-IKKβ), phosphorylated NF-κBp65 (p-NF-κBp65), phos- phorylated p38 (p-p38), and total p38 in the gastrocnemius (GAS) muscle of healthy nontreated mice (n = 5) and cancer-bearing mice treated with vehicle (n = 8) or Scl-Ab (n = 8). Actin was used as loading control. Representative samples are shown. (B) Immunoblot analysis of phosphorylated NF-κBp65 (p-NF-κBp65), phosphorylated p38 (p-p38) and total p38 in C2C12 myoblasts stimulated with vehicle (veh) or TGF-β1. Actin was used as loading control. Representative image of 6 independent experiments is shown. (C) Immunoblot analysis of phosphorylated NF-κBp65, p-p38, and total p38 in C2C12 cells treated with a control peptide or an NF-κB blocking peptide (NPD) and stimulated with vehicle or TGF-β1. Actin was used as loading control. Representative image of 4 independent experiments is shown. (D) Myogenin and MyoD mRNA expression was quantified by qRT-PCR in C2C12 cells after 10 days of myogenic differentiation (n = 4). (E) Pai1 mRNA expression was quantified in the GAS muscle from healthy nontreat- ed mice (n = 5) and from cancer-bearing mice treated with vehicle (n = 8) or Scl-Ab (n = 8). (F) Immunohistochemical staining of Pax7 in the tibialis anterior (TA) muscle from healthy nontreated mice and from mice with bone metastases treated with vehicle or Scl-Ab. Scale bar: 50 μm (top row) and 100 μm (bottom row). (G) Quantification of Pax7-positive cells in the TA muscle from healthy nontreated mice (n = 5) and from mice with bone metastases treated with vehicle (n = 8) or Scl-Ab (n = 8). Data are presented as mean ± SEM. Two-tailed Student’s t test was used to compare 2 groups (D), and ANOVA followed by Tukey’s post hoc analysis was used to compare 3 or more groups (E and G); *P < 0.05, ***P < 0.001.

Article Snippet: MDA-MB-231 cells were transfected with scrambled control siRNA or siRNA against sclerostin (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Control, Blocking Assay, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Two Tailed Test

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: 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: Endocrinology

Article Title: Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence.

doi: 10.1210/en.2015-1244

Figure Lengend Snippet: Figure 1. Hematoxylin/eosin staining of human male bone specimens at 20X magnification (A) and 100X magnification (B) and immunohistochemistry analysis of androgen receptor (AR) expression in decalcified human trabecular bone. C-D) Negative control sample where primary antibodies were omitted. E-F) Double staining for AR (red) and sclerostin (SOST, green). BF bright-field.

Article Snippet: Peptide/protein target Name of Antibody Manufacturer, catalog # Species raised in; monoclonal or polyclonal Dilution used Sclerostin Human SOST/Sclerostin Antibody R&D Systems, AD1406 Goat; polyclonal 1:100 for IHC, 1:500 for WB Alkaline Phosphatase Human Alkaline Phosphatase/ALPL APC-conjugated Antibody R&D Systems, FAB1448A Mouse; monoclonal Undiluted -Actin -Actin (C4) Santa Cruz Biotechnology, Inc, sc47 778 Mouse; monoclonal 1:1000 for WB Androgen Receptor Anti-human Cterminal AR Santa Cruz Biotechnology, sc-815 Rabbit; polyclonal 1:100 for IHC, 1:500 for WB The Endocrine Society.

Techniques: Staining, Immunohistochemistry, Expressing, Negative Control, Double Staining

Journal: Endocrinology

Article Title: Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence.

doi: 10.1210/en.2015-1244

Figure Lengend Snippet: Figure 2. Flow-cytometry assisted cell sorting (FACS) of primary osteocytes from human male bone specimens. A) Gating strategy by morphological features allowed to identify an homogeneous cell population in R1. B) Cells in R1 were evaluated for expression of sclerostin (SOST- FITC) and alkaline phosphatase (ALP-APC). Only SOST/ALP- (SOST) cells were collected and further cultured. SOST-/ALP osteoblasts (ALP) were collected for the assessment of molecular phenotype. C) Sorted SOST osteocytes were further characterized for androgen receptor (AR, green) and sclerostin (SOST, red) by immunofluorescence. Bright-field (BF) images were captured to ascertain the shape of cultured cells. D) RT- PCR was performed assess sclerostin (black bars) and alkaline phosphatase (white bars) gene expression in SOST sorted-osteocytes and ALP

Article Snippet: Peptide/protein target Name of Antibody Manufacturer, catalog # Species raised in; monoclonal or polyclonal Dilution used Sclerostin Human SOST/Sclerostin Antibody R&D Systems, AD1406 Goat; polyclonal 1:100 for IHC, 1:500 for WB Alkaline Phosphatase Human Alkaline Phosphatase/ALPL APC-conjugated Antibody R&D Systems, FAB1448A Mouse; monoclonal Undiluted -Actin -Actin (C4) Santa Cruz Biotechnology, Inc, sc47 778 Mouse; monoclonal 1:1000 for WB Androgen Receptor Anti-human Cterminal AR Santa Cruz Biotechnology, sc-815 Rabbit; polyclonal 1:100 for IHC, 1:500 for WB The Endocrine Society.

Techniques: Flow Cytometry, FACS, Expressing, Cell Culture, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: Endocrinology

Article Title: Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence.

doi: 10.1210/en.2015-1244

Figure Lengend Snippet: Figure 4. Western blot analysis of sclerostin (SOST) gene (panels A and C) and protein (panels B and D) expression in primary cultured osteocytes after 24 hours and 48 hours stimulation with DHT at concentrations ranging from 10-10 M to 10-6 M, compared with unstimulated controls (CTRL). Gene expression data, performed by RT-PCR, are reported as normalized relative quantification (nRQ) referred to -actin as housekeeping gene. Protein expression data, performed by western blot, are reported as the ratio between the band density of SOST with the corresponding band density of -actin (-Act) as housekeeping. Results are the mean of three independent experiments SD. Statistical significance: * P .05; ** P .01; *** P .001. vs. controls.

Article Snippet: Peptide/protein target Name of Antibody Manufacturer, catalog # Species raised in; monoclonal or polyclonal Dilution used Sclerostin Human SOST/Sclerostin Antibody R&D Systems, AD1406 Goat; polyclonal 1:100 for IHC, 1:500 for WB Alkaline Phosphatase Human Alkaline Phosphatase/ALPL APC-conjugated Antibody R&D Systems, FAB1448A Mouse; monoclonal Undiluted -Actin -Actin (C4) Santa Cruz Biotechnology, Inc, sc47 778 Mouse; monoclonal 1:1000 for WB Androgen Receptor Anti-human Cterminal AR Santa Cruz Biotechnology, sc-815 Rabbit; polyclonal 1:100 for IHC, 1:500 for WB The Endocrine Society.

Techniques: Western Blot, Expressing, Cell Culture, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Quantitative Proteomics

Journal: Endocrinology

Article Title: Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence.

doi: 10.1210/en.2015-1244

Figure Lengend Snippet: Figure 5. A-B) Gene (panel A) and protein (panel B) expression of sclerostin (SOST, black bars) and androgen receptor (AR, white bars) in primary cultured osteocytes stimulated with PTH 10-7 M, DHT 10-8 M flutamide (FLUT) 10–6 M and DHT 10–8 M, compared with unstimulated controls (CTRL). Alkaline phosphatase (ALP) gene expression was assessed to confirm molecular phenotype of cultured cells (panel A, gray bars) C-D) Gene (panel C) and protein (panel D) expression of SOST (black bars) and AR (white bars) after siRNA for AR in primary cultured osteocytes stimulated with PTH 10-7 M, DHT 10-8 M FLUT 10-6 M and DHT 10-8 M, compared with unstimulated controls with siRNA for AR (CTRL) and unrelated siRNA provided by the transfection kit (CTRL-). Alkaline phosphatase (ALP) gene expression was assessed to confirm molecular phenotype of cultured cells (panel C, gray bars) Gene expression data, performed by RT-PCR, are reported as normalized relative quantification (nRQ) referred to -actin as housekeeping gene. Data for protein expression are reported as the ratio between the band density of the protein with the corresponding band density of -actin as housekeeping. Results are the mean of three independent experiments SD. Statistical significance: * P .05; ** P .01; P .001 vs. controls.

Article Snippet: Peptide/protein target Name of Antibody Manufacturer, catalog # Species raised in; monoclonal or polyclonal Dilution used Sclerostin Human SOST/Sclerostin Antibody R&D Systems, AD1406 Goat; polyclonal 1:100 for IHC, 1:500 for WB Alkaline Phosphatase Human Alkaline Phosphatase/ALPL APC-conjugated Antibody R&D Systems, FAB1448A Mouse; monoclonal Undiluted -Actin -Actin (C4) Santa Cruz Biotechnology, Inc, sc47 778 Mouse; monoclonal 1:1000 for WB Androgen Receptor Anti-human Cterminal AR Santa Cruz Biotechnology, sc-815 Rabbit; polyclonal 1:100 for IHC, 1:500 for WB The Endocrine Society.

Techniques: Expressing, Cell Culture, Gene Expression, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative Proteomics

Journal: Endocrinology

Article Title: Regulation of Sclerostin Production in Human Male Osteocytes by Androgens: Experimental and Clinical Evidence.

doi: 10.1210/en.2015-1244

Figure Lengend Snippet: Figure 6. Correlation between serum sclerostin and total testosterone in the control group (CTRL, straight line, n 20) and in hypogonadal patients (HP, dotted line, n 20).

Article Snippet: Peptide/protein target Name of Antibody Manufacturer, catalog # Species raised in; monoclonal or polyclonal Dilution used Sclerostin Human SOST/Sclerostin Antibody R&D Systems, AD1406 Goat; polyclonal 1:100 for IHC, 1:500 for WB Alkaline Phosphatase Human Alkaline Phosphatase/ALPL APC-conjugated Antibody R&D Systems, FAB1448A Mouse; monoclonal Undiluted -Actin -Actin (C4) Santa Cruz Biotechnology, Inc, sc47 778 Mouse; monoclonal 1:1000 for WB Androgen Receptor Anti-human Cterminal AR Santa Cruz Biotechnology, sc-815 Rabbit; polyclonal 1:100 for IHC, 1:500 for WB The Endocrine Society.

Techniques: Control