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: The Journal of Cell Biology

Article Title: Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

doi: 10.1083/jcb.201007141

Figure Lengend Snippet: Kindlin-3 −/− mice develop severe osteopetrosis. (A and B) Histology of tibiae of P4 wild-type and kindlin-3 −/− mice stained with hematoxylin and eosin (A) and van Kossa (B). (C and D) Quantitative peripheral computer tomography measurements determining bone mineral density (C) and histomorphometric analysis to determine bone surface from bones of P4 wild-type and kindlin-3 −/− mice (D); n = 3. (E) Histology of tibiae of P4 wild-type and kindlin-3 −/− mice stained for TRAP activity. (F and G) Histomorphometric analyses determining the number of osteoclasts (F) and surface covered by osteoclasts (G) of wild-type and kindlin-3 −/− mice; n = 3. (H) Ca 2+ levels in serum of P4 wild-type and kindlin-3 −/− mice; n = 8. (I) PTH levels in plasma of P3 wild-type and kindlin-3 −/− mice; n = 10. (J and K) Resorption pits (J) and their quantification (K) of wild-type and kindlin-3 −/− osteoclasts cultured on calcium apatite coated slides; n = 7. (L) Zymography of cell culture supernatants from primary wild-type and kindlin-3 −/− osteoclasts. (M) Cathepsin K activity from lysates of primary wild-type and kindlin-3 −/− osteoclasts; n = 11. Data are presented as mean ± SD (error bars). P-values indicate significant differences from wild-type (Student’s t test). n.s., not significant. Bars: (A and B) 250 µm; (E, top) 250 µm; (E, bottom) 25 µm; (J) 100 µm.

Article Snippet: Nonadherent cells were collected after 24 h. Leukocytes were isolated from the interface after centrifugation at 1,000 g for 20 min in leukocyte separation medium (Laboratoires Eurobio), then washed with α-MEM medium and seeded at a concentration of 2,000 cells/mm 2 in osteoclast differentiation medium (α-MEM containing 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin with 50 ng/ml M-CSF and 40 ng/ml RANKL).

Techniques: Staining, Tomography, Activity Assay, Clinical Proteomics, Cell Culture, Zymography

Journal: The Journal of Cell Biology

Article Title: Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

doi: 10.1083/jcb.201007141

Figure Lengend Snippet: Differentiation of kindlin-3 −/− osteoclasts. (A) RANKL/OPG ratio in plasma of P3 wild-type and kindlin-3 −/− mice; n = 11. (B) RT-PCR of osteoclastogenic markers upon M-CSF and RANKL treatment of wild-type and kindlin-3 −/− fetal liver cells. (C) RT-PCR of kindlin-1 and -2 expression during in vitro osteoclast differentiation. RNA from keratinocytes served as positive control. (D) Number of nuclei per osteoclast 5 d after induction of differentiation; 1,004 cells of each genotype obtained from five independent experiments were analyzed. (E) Osteoclast nuclear numbers determined from histological sections of P4 tibiae. 63 and 252 osteoclasts from wild-type and kindlin-3 −/− bone sections were analyzed, respectively. Data are presented as mean ± SD (error bars). P-values indicate significant differences from wild-type (Student’s t test).

Article Snippet: Nonadherent cells were collected after 24 h. Leukocytes were isolated from the interface after centrifugation at 1,000 g for 20 min in leukocyte separation medium (Laboratoires Eurobio), then washed with α-MEM medium and seeded at a concentration of 2,000 cells/mm 2 in osteoclast differentiation medium (α-MEM containing 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin with 50 ng/ml M-CSF and 40 ng/ml RANKL).

Techniques: Clinical Proteomics, Reverse Transcription Polymerase Chain Reaction, Expressing, In Vitro, Positive Control

Journal: The Journal of Cell Biology

Article Title: Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

doi: 10.1083/jcb.201007141

Figure Lengend Snippet: Integrin defects in kindlin-3 −/− osteoclasts. (A) Adhesion of primary wild-type and kindlin-3 −/− osteoclasts to osteopontin. Number of adherent cells per field of view (FOV) is shown. (B) Surface expression of β1, β3, β5, and αV integrins on wild-type (green) and kindlin-3 −/− (red) macrophages. Isotype control staining is shown in dark blue. Numbers above graphs indicate integrin expression on kindlin-3 −/− cells (mean ± SD) relative to wild-type cells ( n = 6). (C) 9EG7 binding on wild-type and kindlin-3 −/− macrophages in the presence or absence of 2 mM MnCl 2 . (D) Binding of Alexa Fluor 647–labeled FNIII7-10 by wild-type and kindlin-3 −/− macrophages in the presence or absence of 3 mM MnCl 2 . Data show mean ± SD of four independent experiments and were subtracted by background binding of the isotype and EDTA control, respectively. (E) Wild-type and kindlin-3 −/− macrophages plated on ICAM-1 in the presence or absence of 1 mM MnCl 2 . P-values indicate significant differences from wild-type (Student’s t test).

Article Snippet: Nonadherent cells were collected after 24 h. Leukocytes were isolated from the interface after centrifugation at 1,000 g for 20 min in leukocyte separation medium (Laboratoires Eurobio), then washed with α-MEM medium and seeded at a concentration of 2,000 cells/mm 2 in osteoclast differentiation medium (α-MEM containing 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin with 50 ng/ml M-CSF and 40 ng/ml RANKL).

Techniques: Expressing, Control, Staining, Binding Assay, Labeling

Journal: The Journal of Cell Biology

Article Title: Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

doi: 10.1083/jcb.201007141

Figure Lengend Snippet: Defective spreading of kindlin-3 −/− osteoclasts. (A) Wild-type and kindlin-3 −/− osteoclasts grown on glass coverslips and stained for TRAP. (B) F-actin (stained with phalloidin, green) and nuclei (DAPI, blue) in wild-type and kindlin-3 −/− osteoclasts. Bars, 100 µm.

Article Snippet: Nonadherent cells were collected after 24 h. Leukocytes were isolated from the interface after centrifugation at 1,000 g for 20 min in leukocyte separation medium (Laboratoires Eurobio), then washed with α-MEM medium and seeded at a concentration of 2,000 cells/mm 2 in osteoclast differentiation medium (α-MEM containing 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin with 50 ng/ml M-CSF and 40 ng/ml RANKL).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

doi: 10.1083/jcb.201007141

Figure Lengend Snippet: Impaired adhesion and M-CSF signaling in kindlin-3 −/− pre-osteoclasts. (A) Wild-type and kindlin-3 −/− pre-osteoclasts either maintained in suspension (S) or replated on vitronectin (A). Western blotting for kindlin-3, p-FAK, FAK, p-src, Src, p-Pyk2, and Pyk2. GAPDH and actin served as loading controls. (B) TRAP staining of wild-type and kindlin-3 −/− osteoclasts treated with 40 ng/ml RANKL together with either 20 ng/ml or 100 ng/ml M-CSF. Bar, 250 µm. (C) Starved wild-type and kindlin-3 −/− osteoclasts treated with either 10 ng/ml or 100 ng/ml M-CSF. Western blotting for activated Erk and Akt. Activated Syk was determined by immunoprecipitation followed by immunoblotting with anti-phosphotyrosine (4G10) antibody.

Article Snippet: Nonadherent cells were collected after 24 h. Leukocytes were isolated from the interface after centrifugation at 1,000 g for 20 min in leukocyte separation medium (Laboratoires Eurobio), then washed with α-MEM medium and seeded at a concentration of 2,000 cells/mm 2 in osteoclast differentiation medium (α-MEM containing 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin with 50 ng/ml M-CSF and 40 ng/ml RANKL).

Techniques: Suspension, Western Blot, Staining, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

doi: 10.1083/jcb.201007141

Figure Lengend Snippet: Impaired podosome formation in kindlin-3 −/− osteoclasts. (A) Vinculin (red) and F-actin staining (phalloidin, green) of pre-osteoclasts plated on glass coverslips and observed by confocal microscopy. (B) Fluorescence intensity profile through three actin-core units (indicated by the white lines in A) of wild-type and kindlin-3 −/− pre-osteoclasts. (C and D) vinculin (red) and F-actin staining (phalloidin, blue) together with αv integrin (green; C) or β1 integrin (green; D). Bars (A and C) 5 µm; (D) 10 µm.

Article Snippet: Nonadherent cells were collected after 24 h. Leukocytes were isolated from the interface after centrifugation at 1,000 g for 20 min in leukocyte separation medium (Laboratoires Eurobio), then washed with α-MEM medium and seeded at a concentration of 2,000 cells/mm 2 in osteoclast differentiation medium (α-MEM containing 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin with 50 ng/ml M-CSF and 40 ng/ml RANKL).

Techniques: Staining, Confocal Microscopy, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

doi: 10.1083/jcb.201007141

Figure Lengend Snippet: Podosomal actin core formation is not abolished in kindlin-3 −/− cells. (A) Scanning electron microscopy of basal membrane preparations of wild-type and kindlin-3 −/− osteoclasts. (B–D) Colocalization of cortactin (red) and F-actin (phalloidin, green; B), F-actin (red) and Arp2/3 (green; C), and WASp (red) and F-actin (green; D) in podosome clusters of wild-type and kindlin-3 −/− pre-osteoclasts analyzed by confocal microscopy. Bars: (A, left) 1 µm; (A, right) 500 nm; (B–D) 10 µm.

Article Snippet: Nonadherent cells were collected after 24 h. Leukocytes were isolated from the interface after centrifugation at 1,000 g for 20 min in leukocyte separation medium (Laboratoires Eurobio), then washed with α-MEM medium and seeded at a concentration of 2,000 cells/mm 2 in osteoclast differentiation medium (α-MEM containing 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin with 50 ng/ml M-CSF and 40 ng/ml RANKL).

Techniques: Electron Microscopy, Membrane, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

doi: 10.1083/jcb.201007141

Figure Lengend Snippet: Abnormal F-actin distribution but normal microtubule organization and acetylation in kindlin-3 −/− osteoclasts. (A) Vinculin (red) and F-actin staining (phalloidin, green) of wild-type and kindlin-3 −/− osteoclasts plated on glass coverslips. (B) Vinculin (red) and F-actin staining (phalloidin, green) of wild-type and kindlin-3 −/− osteoclasts plated on mineral surface of osteologic slides. DAPI (blue) shows nuclei (A and B). (C) Wild-type and kindlin-3 −/− osteoclasts were labeled with phalloidin (blue), anti-acetylated tubulin (green), and anti-tubulin antibodies. (D) Cell lysates from wild-type and kindlin-3 −/− osteoclasts were immunoblotted with antibodies against kindlin-3, acetylated tubulin, and tubulin. Antibodies against actin and GAPDH were used as loading controls. Bars: (A) 25 µm; (B) 100 µm; (C) 10 µm.

Article Snippet: Nonadherent cells were collected after 24 h. Leukocytes were isolated from the interface after centrifugation at 1,000 g for 20 min in leukocyte separation medium (Laboratoires Eurobio), then washed with α-MEM medium and seeded at a concentration of 2,000 cells/mm 2 in osteoclast differentiation medium (α-MEM containing 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin with 50 ng/ml M-CSF and 40 ng/ml RANKL).

Techniques: Staining, Labeling

Journal: The Journal of Cell Biology

Article Title: Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

doi: 10.1083/jcb.201007141

Figure Lengend Snippet: Podosomes and actin belts in integrin-deficient osteoclasts. (A) Wild-type and single, double, and triple integrin-deficient osteoclasts stained for TRAP. (B) Vinculin (red) and F-actin staining (phalloidin, green) of wild-type and integrin-deficient pre-osteoclasts plated on glass. (C) Diameters of actin dots in pre-osteoclasts with indicated genotypes measured using MetaMorph software; n = 6/5/5/8/9/6/6/6/8/9 different cells from each genotype taken to measure the actin core size. (D) Vinculin (red) and F-actin staining (phalloidin, green) of wild-type and integrin-deficient osteoclasts plated on glass. DAPI (blue) shows nuclei. (E) Diameter of podosomal belts of wild-type and integrin-deficient osteoclasts plated on glass. Data are presented as mean ± SD (error bars). P-values indicate significant differences from wild-type (Student’s t test). Bars: (A) 100 µm; (B) 2 µm; (D, top) 25 µm; (D, bottom) 5 µm.

Article Snippet: Nonadherent cells were collected after 24 h. Leukocytes were isolated from the interface after centrifugation at 1,000 g for 20 min in leukocyte separation medium (Laboratoires Eurobio), then washed with α-MEM medium and seeded at a concentration of 2,000 cells/mm 2 in osteoclast differentiation medium (α-MEM containing 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin with 50 ng/ml M-CSF and 40 ng/ml RANKL).

Techniques: Staining, Software

Journal: The Journal of Cell Biology

Article Title: Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

doi: 10.1083/jcb.201007141

Figure Lengend Snippet: Sealing zones of integrin-deficient osteoclasts. (A) Vinculin (red) and F-actin staining (phalloidin, green) of wild-type and integrin-deficient osteoclasts plated on a mineral surface. DAPI staining (blue) shows nuclei. Bar, 25 µm. (B) Resorption activity of wild-type and integrin-deficient osteoclasts plated on calcium apatite-coated slides quantified with MetaMorph. Number of analyzed slides per genotype: n = 26/6/6/6/6/3/3/3/8. Data are presented as mean ± SD (error bars). P-values indicate significant differences from wild-type (Student’s t test).

Article Snippet: Nonadherent cells were collected after 24 h. Leukocytes were isolated from the interface after centrifugation at 1,000 g for 20 min in leukocyte separation medium (Laboratoires Eurobio), then washed with α-MEM medium and seeded at a concentration of 2,000 cells/mm 2 in osteoclast differentiation medium (α-MEM containing 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin with 50 ng/ml M-CSF and 40 ng/ml RANKL).

Techniques: Staining, Activity Assay