Journal: Frontiers in Endocrinology

Article Title: Calcitonin Induces Bone Formation by Increasing Expression of Wnt10b in Osteoclasts in Ovariectomy-Induced Osteoporotic Rats

doi: 10.3389/fendo.2020.00613

Figure Lengend Snippet: Calcitonin treatment increases the osteoclastic Wnt10b expression in metaphysis of femoral bone in ovariectomy-induced osteoporotic rats. Sham-operated rats and ovariectomized (OVX) rats were treated as indicated in . Immunohistochemistry labeling of Wnt10b (green) and TRAP stain (red) were performed near resorption zone of growth plate in femoral bone. Decreased Wnt10b expression was found in OVX rats. CT treatment increased Wnt10b expression. The number of the osteoblast, osteoclast, and osteoclast with Wnt10b were counted. Green arrow showed osteoclasts to secret Wnt10b; White arrow showed osteoclasts that did not secret Wnt10b. Scale bar = 50 μm. *Indicates a significant difference ( p < 0.05). N = 6 in each group.

Article Snippet: A total of 10 6 harvested cells were cultured in a 10 cm dish with osteoclast differentiation medium (α-MEM medium containing 10% fetal bovine serum (FBS, SH30088.03, GE Healthcare, USA), 50 ng/mL macrophage colony-stimulating factor (M-CSF, 400-28, PEPROTECH, Rocky Hill, NJ, USA), 50 ng/mL RANKL (315-11, PEPROTECH, USA), and 1% Antibiotic-Antimycotic Solution (30-004-CI, CORNING, Corning, NY, USA).

Techniques: Expressing, Immunohistochemistry, Labeling, Staining

Journal: Frontiers in Endocrinology

Article Title: Calcitonin Induces Bone Formation by Increasing Expression of Wnt10b in Osteoclasts in Ovariectomy-Induced Osteoporotic Rats

doi: 10.3389/fendo.2020.00613

Figure Lengend Snippet: Calcitonin increases intracellular Wnt10b expression in osteoclasts. Bone marrow monocytes were isolated from femoral and tibial bones of 8-week-old SD rats. They were induced into osteoclasts by M-CSF and RANKL stimulation. (A) Confocal analysis was performed in osteoclasts treated with 3 nM calcitonin alone or with C59 for 16 h. Osteoclasts were labeled with rhodamine phalloidin (red) to visualize F-actin and Nuclear Red (blue) to visualize nuclei. Scale bar = 20 μm. (B) The statistics of Wnt10b fluorescent intensity was showed in each group. *Indicates a significant difference ( p < 0.05).

Article Snippet: A total of 10 6 harvested cells were cultured in a 10 cm dish with osteoclast differentiation medium (α-MEM medium containing 10% fetal bovine serum (FBS, SH30088.03, GE Healthcare, USA), 50 ng/mL macrophage colony-stimulating factor (M-CSF, 400-28, PEPROTECH, Rocky Hill, NJ, USA), 50 ng/mL RANKL (315-11, PEPROTECH, USA), and 1% Antibiotic-Antimycotic Solution (30-004-CI, CORNING, Corning, NY, USA).

Techniques: Expressing, Isolation, Labeling

Journal: Frontiers in Endocrinology

Article Title: Calcitonin Induces Bone Formation by Increasing Expression of Wnt10b in Osteoclasts in Ovariectomy-Induced Osteoporotic Rats

doi: 10.3389/fendo.2020.00613

Figure Lengend Snippet: Calcitonin increases Wnt10b levels in osteoclasts. Osteoclasts were prepared and treated as indicated in . Western blot analysis was performed on osteoclasts treated with 3 nM calcitonin alone or with C59 for the various times indicated. Protein levels were quantified by densitometry, corrected for sample loading on the basis of actin levels, and expressed as the fold change relative to the control lane. Each blot is representative of at least three replicate experiments.

Article Snippet: A total of 10 6 harvested cells were cultured in a 10 cm dish with osteoclast differentiation medium (α-MEM medium containing 10% fetal bovine serum (FBS, SH30088.03, GE Healthcare, USA), 50 ng/mL macrophage colony-stimulating factor (M-CSF, 400-28, PEPROTECH, Rocky Hill, NJ, USA), 50 ng/mL RANKL (315-11, PEPROTECH, USA), and 1% Antibiotic-Antimycotic Solution (30-004-CI, CORNING, Corning, NY, USA).

Techniques: Western Blot, Control

Journal: Frontiers in Endocrinology

Article Title: Calcitonin Induces Bone Formation by Increasing Expression of Wnt10b in Osteoclasts in Ovariectomy-Induced Osteoporotic Rats

doi: 10.3389/fendo.2020.00613

Figure Lengend Snippet: Model of calcitonin-induced bone formation through action on osteoclasts. Bones undergo constant remodeling throughout the lifetime of the organism, and this involves the continuous activity of osteoblasts and osteoclasts. Osteoblastic bone formation is communicated to osteoclastic bone resorption by positive and negative modulators (RANKL/OPG or WNT). Osteoclasts also communicate to osteoblasts by clastokines (Wnt10b and S1P). Bone anabolic such as PTH also increase osteoclastic bone resorption through their control of RANKL/OPG and WNT signaling. Anti-resorptive inhibit osteoclast resorption, which usually causes the inhibition of bone formation. Uncoupling anti-resorptive, such as calcitonin, may modulate osteoblast mineralization through the controlled secretion of Wnt10b and S1P.

Article Snippet: A total of 10 6 harvested cells were cultured in a 10 cm dish with osteoclast differentiation medium (α-MEM medium containing 10% fetal bovine serum (FBS, SH30088.03, GE Healthcare, USA), 50 ng/mL macrophage colony-stimulating factor (M-CSF, 400-28, PEPROTECH, Rocky Hill, NJ, USA), 50 ng/mL RANKL (315-11, PEPROTECH, USA), and 1% Antibiotic-Antimycotic Solution (30-004-CI, CORNING, Corning, NY, USA).

Techniques: Activity Assay, Control, Inhibition

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: Mice were treated as described in Figure 2. a.) Representative images of 5 μm slices of distal femur stained for TRAP (20x magnification)(red shading; n=4 Vh, 5 TBT). b.) Oc/B.Per.: number of TRAP-positive osteoclasts per nm of trabecular perimeter, excluding cortex. Trab. Perimeter: total perimeter of trabecular bone (um). c.) Quantification of serum TRAP by ELISA (n = 12 Vh, 11 TBT 10 mg/kg). Whole humerus bone mRNA expression (n = 12 Vh, 11 TBT 10 mg/kg) of d.) Osteoclast differentiation markers Nfatc1, Apc5 and Ctsk, and e.) Intercellular communication proteins Rankl/Opg (osteoblast to osteoclast), Ct1 and Sema4d (osteoclast to osteoblast). Data are presented as mean ± SE. *p < 0.05 **p < 0.01, Mann-Whitney.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing, MANN-WHITNEY

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: Primary bone marrow macrophages were isolated from female C57BL/6J mice, induced to differentiate to osteoclasts with M-CSF and RANKL (see Methods), and after 24 hrs treated with Vh (DMSO), TBT (20, 50, or 80 nM), rosiglitazone (Rosi, 500 nM, PPARγ agonist), LG100268 (LG268, RXR agonist 1 μM), or T0101317 (T317, LXRα/β agonist, 1 μM) for a total of 6 days of differentiation. a.) mRNA expression of osteoclast differentiation markers. n=10–17 independent cultures. Data are presented as mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001 compared to Vh, one-way ANOVA (Dunnett’s). TBT treatments were compared to Vh separately from control comparisons. b.) Representative images of TRAP-positive, multinucleated (3 or more nuclei) cells (Scale bar = 400 μm) n=10 independent cultures. c.) Representative images of von Kossa stained mineral surface after 3 days of incubation with differentiated osteoclasts. n=5 independent cultures.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Isolation, Expressing, Control, Staining, Incubation

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: a.) Mice were treated as described in Figure 2. Whole humerus bone mRNA expression of LXR-dependent genes Abca1 and Srebp1c. Data are presented as mean ± SE. n = 11–12 individual mice. **p<0.01, Mann-Whitney b.) Osteoclast cultures were prepared as described in Figure 5 and treated with Vh (DMSO), TBT (20, 50, or 80 nM), rosiglitazone (Rosi, 500 nM, PPARγ agonist), LG100268 (LG268, RXR agonist 1 μM), or T0101317 (T317, LXRα/β agonist, 1 μM) and analyzed for mRNA expression Abca1 and Srebp1c. n=10–17 independent cultures. Data are presented as mean ± SE. *p<0.05, **p<0.01, ***p<0.001 compared to Vh, one-way ANOVA (Dunnett’s). TBT treatments were compared to Vh separately from control comparisons. c.) Primary bone marrow macrophages were isolated from male and female LXRα +/+, +/− and −/− mice, induced to differentiate into osteoclasts with M-CSF and RANKL, treated after 24 hrs with Vh (DMSO) or TBT (50 nM) for 4 days, and analyzed for mRNA expression of Ctsk. n=4 independent cultures. Data are presented as mean ± SE. Two-way ANOVA. d.) Osteoclast cultures were prepared as described in b and treated with Vh (DMSO) or TBT (50 nM) with or without GSK2033 (GSK, LXR antagonist, 0.8 μM) and analyzed for mRNA expression Abca1, Srebp1c and Ctsk. n=5 independent cultures. Data are presented as mean ± SE. Two-way ANOVA.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Expressing, MANN-WHITNEY, Control, Isolation

Journal: Journal of cellular physiology

Article Title: Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice

doi: 10.1002/jcp.26495

Figure Lengend Snippet: a.) Mice were treated as described in Figure 2. Whole humerus bone mRNA expression the RXR-dependent gene Mafb. Data are presented as mean ± SE. n = 11–12 individual mice. **p<0.01, Mann-Whitney b.) Osteoclast cultures were prepared as described in Figure 5 and treated with Vh (DMSO), TBT (20, 50, or 80 nM), rosiglitazone (500 nM), LG100268 (LG268, 1 μM), or T0101317 (T317, LXRα/β agonist, 1 μM) and analyzed for mRNA expression of Mafb. Data are presented as mean ± SE. n=10–17 independent cultures. *p<0.05, **p<0.01, *** p<0.001 compared to Vh, one-way ANOVA (Dunnett’s). TBT treatments were compared to Vh separately from control comparisons. Osteoclast cultures were prepared as described in b and treated with Vh (DMSO) or TBT (50 nM) with or without HX531 (RXR antagonist, 1 μM) and analyzed for mRNA expression Mafb and Ctsk. n=5 independent cultures. Data are presented as mean ± SE. Two-way ANOVA.

Article Snippet: For resorption assays, cells were collected from the differentiating osteoclast cultures, counted, plated (40,000 per well in 800 μl osteoclast differentiation medium) in 24-well Corning Osteo-Assay plates (CLS3987, Sigma Aldrich) and re-treated with Vh or TBT.

Techniques: Expressing, MANN-WHITNEY, Control

Journal: Stem Cells International

Article Title: Mesenchymal Progenitors Derived from Different Locations in Long Bones Display Diverse Characteristics

doi: 10.1155/2019/5037578

Figure Lengend Snippet: Conditioned medium from E-MPs promoted osteoclast formation. (a) TRAP staining was used to detect osteoclast formation from osteoclast precursors, after culturing in osteoclastic differentiation medium with CM from different MPs for 7 days. Scale bar, 200 μ m. OCi: osteoclast induction. (b) TRAP-positive cells (nuclei ≥ 3) were counted. (c) The mRNA levels of osteoclast marker genes were detected by qRT-PCR. (d) The expression of several paracrine genes which were related to osteoclast formation was evaluated in the three MP groups. Data was shown as mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Osteoclast precursors were incubated with osteoclastic differentiation medium ( α -MEM with 10% FBS, 50 ng/ml M-CSF, and 100 ng/ml RANKL (PeproTech)) for 7 days to induce maturation into osteoclasts.

Techniques: Staining, Marker, Quantitative RT-PCR, Expressing