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: 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: 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: International Journal of Molecular Medicine

Article Title: Icaritin promotes the osteogenesis of bone marrow mesenchymal stem cells via the regulation of sclerostin expression

doi: 10.3892/ijmm.2020.4470

Figure Lengend Snippet: Icaritin (1 µ M) increases mRNA levels of osteogenic marker genes. (A) SOST , (B) OCN , (C) Runx2 , (D) Alp and (E) β-catenin at the different time points of osteogenesis of human bone marrow-derived mesenchymal stem cells. DMSO was used as the control group. Data are presented as the mean ± standard deviation (n=3). * P<0.05 and ** P<0.01 vs. control group at the same stage. SOST, sclerostin; OCN, osteocalcin; Runx2, RUNX family transcription factor 2; Alp, alkaline phosphatase.

Article Snippet: The membrane was incubated with antibodies targeting β-actin (1:1,000; cat. no. 4970S; Cell Signaling Technology, Inc.), osteocalcin (OCN; 1:1,000; cat. no. MAB1419; R&D Systems, Inc.), RUNX family transcription factor 2 (Runx2; 1:1,000; cat. no. 12556; Cell Signaling Technology, Inc.), ALP (1:1,000; cat. no. AF2910; R&D Systems, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc.) and SOST (1:1,000; cat. no. MAB1406; R&D Systems, Inc.) overnight at 4°C.

Techniques: Marker, Derivative Assay, Control, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Icaritin promotes the osteogenesis of bone marrow mesenchymal stem cells via the regulation of sclerostin expression

doi: 10.3892/ijmm.2020.4470

Figure Lengend Snippet: SOST overexpression reverses icaritin-induced osteogenesis of hBMSCs. (A) Western blot analysis for the protein level of SOST. (B) The mRNA expression of SOST in BMSCs, BMSCs-vector, and BMSCs-SOST. (C) Mineralization in cultured hBMSCs in BMSCs-vector and BMSCs-SOST groups with or without icaritin were detected at day 14. Magnification, ×10. (D) The mRNA levels of OCN, Runx2, Alp and β-actin were determined by reverse transcription quantitative polymerase chain reaction. Data are presented as mean ± standard deviation (n=3). ** P<0.01 vs. BMSCs group; # P<0.05 and ## P<0.01 vs. BMSCs-vector group. SOST, sclerostin; BMSCs, bone marrow-derived mesenchymal stem cells; OCN, osteocalcin; Runx2, RUNX family transcription factor 2; Alp, alkaline phosphatase.

Article Snippet: The membrane was incubated with antibodies targeting β-actin (1:1,000; cat. no. 4970S; Cell Signaling Technology, Inc.), osteocalcin (OCN; 1:1,000; cat. no. MAB1419; R&D Systems, Inc.), RUNX family transcription factor 2 (Runx2; 1:1,000; cat. no. 12556; Cell Signaling Technology, Inc.), ALP (1:1,000; cat. no. AF2910; R&D Systems, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc.) and SOST (1:1,000; cat. no. MAB1406; R&D Systems, Inc.) overnight at 4°C.

Techniques: Over Expression, Western Blot, Expressing, Plasmid Preparation, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Derivative Assay