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: 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

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