Journal: Bone Reports

Article Title: GLP-2 administration in ovariectomized mice enhances collagen maturity but did not improve bone strength

doi: 10.1016/j.bonr.2020.100251

Figure Lengend Snippet:

Article Snippet: Soluble murine RANKL , Bio-Techne , 462-TEC-010.

Techniques: Electron Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Microbiota-Derived Metabolites, Indole-3-aldehyde and Indole-3-acetic Acid, Differentially Modulate Innate Cytokines and Stromal Remodeling Processes Associated with Autoimmune Arthritis

doi: 10.3390/ijms22042017

Figure Lengend Snippet: IAld enhanced the bone remodeling process of osteoclastogenesis. RAW cells were differentiated with receptor activator of NF-κB-ligand (RANKL) alone or with RANKL in the presence of either IAld or I3AA (0.0125–0.2 mM) as described in Methods. ( a , b ) Cells were fixed and then stained for tartrate-acid phosphates (TRAP) expression on d 5 and the number of osteoclast-like cells were enumerated from multiple regions of each well ( n = 5). Data was reported as the percentage of osteoclast-like cells relative to the RANKL-treated controls (Mean ± SEM) from a representative experiment. Statistical difference relative to RANKL-treated control cells was determined by ordinary one-way ANOVA with post-hoc test for multiple comparisons. ( c , d ) The transcription of osteoclast genes TRAP and CatK on d 5 is reported from the combined results (Fold Induction) of 3 independent experiments ( n = 3). Statistical difference was determined by paired Student’s t test. Indicators of statistical difference Two-tailed p -value: NS = Not significant, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. ( e ) Representative 20× magnification images taken from d 5 TRAP-stained cells: untreated, RANKL-treated control and cells treated with RANKL in the presence of IAld (0.05 mM) or I3AA (0.2 mM). Osteoclast-like cells are indicated by a red arrow. All results depict consistent trends observed in multiple independent experiments.

Article Snippet: Recombinant human vascular endothelial growth factor peptide (VEGF 165 ) and murine soluble receptor activated NF-κB ligand (RANKL) was supplied by Peprotech (Rockhill, NJ, USA).

Techniques: Staining, Expressing, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Microbiota-Derived Metabolites, Indole-3-aldehyde and Indole-3-acetic Acid, Differentially Modulate Innate Cytokines and Stromal Remodeling Processes Associated with Autoimmune Arthritis

doi: 10.3390/ijms22042017

Figure Lengend Snippet: AhR antagonist CH-223191 inhibited the disruption of angiogenesis by I3AA but failed to alter the effect of IAld on angiogenesis and osteoclastogenesis. ( a , b ) HUVEC were seeded onto Matrigel with VEGF (25 ng/mL). After 1 h, cells were additionally treated with either IAld (0.5 mM) ( a ), I3AA (0.5 mM) ( b ), CH-223191 (3 μM) or a combination as indicated. The percentage of loops relative to VEGF-treated control cells (Veh) is reported ( n = 8). ( c , d ) RAW cells were treated RANKL alone or RANKL in addition with IAld (0.05 mM) ( c ), I3AA (d), CH-223191 (10 μM) or a combination as indicated. The percentage of osteoclast-like cells relative to RANKL-treated control cells (Veh) was reported ( n = 5). Statistical difference was determined on unnormalized data of either the number of loops formed ( a , b ) or from the number of osteoclast-like cells ( c , d ) by Ordinary one-way ANOVA with post-hoc test for multiple comparisons. Indicators of statistical different are between the respective controls (Veh) of the experimental type and the treatment group for which the mark is above, unless otherwise indicated by a bar. NS = not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data are from one experiment representative of several separate experiments.

Article Snippet: Recombinant human vascular endothelial growth factor peptide (VEGF 165 ) and murine soluble receptor activated NF-κB ligand (RANKL) was supplied by Peprotech (Rockhill, NJ, USA).

Techniques: Disruption, Control

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a Immunofluorescence staining of BRD9 (red) and CTSK (green) in the mouse distal femur at 4 weeks of age. White dashed lines outline the surface of trabecular bone. Scale bar, 100 μm. b Schema for osteoclastic differentiation stages of BMDMs in vitro. Bone marrow-derived monocytes, BMDMs, osteoclast precursors, preOCs, and matured osteoclast, OCs. c Brd9 mRNA expression in BMDMs at indicated days after RANKL-induction, as measured by qPCR. n = 3 biologically independent samples. d BRD9, MMP9, CTSK and ACP5 protein expression in BMDMs at indicated days after RANKL-induction. e Immunofluorescence staining of BRD9 (green) and TRAP staining (red) in BMDMs after 3 days of RANKL-induction. Colored boxes in the first panel are shown at higher magnification in below. Dashed lines outline the cell boundaries. Scale bar, 100 μm. f Representative micro-CT image of the distal trabecular bone and cortical bone of femurs from 4-week-old LysM-Cre;Brd9 fl/fl mice and littermate control mice. Arrows indicates the decreased bone mass. Scale bar, 2 mm. g Quantification analysis of bone volume/tissue volume ratio (BV/TV), bone surface/volume ratio (BS/BV), bone volume/tissue volume ratio (BS/TV), porosity percent (Po), trabecular thickness (Tb. Th), trabecular number (Tb. N), trabecular separation (Tb. Sp), bone mineral density (BMD), cortical crossectional bone area (Ct. Ar) and perimeter (Ct. Pm) of femurs from 4-week-old LysM-Cre;Brd9 fl/fl mice and littermate control mice. n = 3. h H&E staining of femurs from 4-week-old LysM-Cre;Brd9 fl/fl mice and littermate control mice. Box in the first panel is shown at higher magnification in below. Asterisk indicates the decreased bone mass. Scale bar, 200 μm. i Von Kossa staining of femurs from 4-week-old LysM-Cre;Brd9 fl/fl mice and littermate control mice. Box in the first panel is shown at higher magnification in below. Asterisk indicates the decreased bone mineralization. Scale bar, 200 μm. All data in this figure are represented as mean ± SD. Two-tailed Student’s t -test for ( c ) and ( g ). All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For osteoclast induction, BMDMs were cultured into complete media with 50 ng/ml recombinant soluble murine M-CSF (PeproTech, 315-02) and 100 ng/ml recombinant soluble murine RANKL (PeproTech, 315-11).

Techniques: Immunofluorescence, Staining, In Vitro, Derivative Assay, Expressing, Micro-CT, Control, Two Tailed Test

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a TRAP staining and quantification analysis of BMDMs from 4-week-old LysM-Cre;Brd9 fl/fl mice and littermate control mice after 3 days of RANKL-induction. Scale bar, 200 μm. n = 8. b The mRNA expression of osteoclastic-specific genes of Acp5 , Ctsk, and Mmp9 in BMDMs from LysM-Cre;Brd9 fl/fl mice compared with that from control littermates after 3 days of RANKL-induction, as measured by qPCR. n = 3 for LysM-Cre;Brd9 fl/fl mice group. n = 6 for control littermates. c The protein expression of MMP9 and CTSK in BMDMs from LysM-Cre;Brd9 fl/fl mice compared with that from control littermates after 3 days of RANKL-induction, as measured by western blot. d Cell viability of RANKL-induced BMDMs with 1 day of iBRD9 at different concentration, shown by cell counting kit 8 assay. n = 10 biologically independent samples. e The mRNA expression of Acp5 and Mmp9 in RANKL-induced BMDMs with 1 day of iBRD9 at different concentration, as measured by qPCR. n = 5 biologically independent samples for Acp5 . n = 3 biologically independent samples for Mmp9 . f TRAP staining and quantification analysis of RANKL-induced BMDMs with 5 days of iBRD9 at different concentration. Scale bar, 200 μm. n = 5 biologically independent samples. g The protein expression of FOS and CTSK in BMDMs after 3 days of 1 μM iBRD9 or vector treatment with or without RANKL-induction, as measured by western blot. M-CSF, M; RANKL, R. h The mRNA expression of Acp5 and Mmp9 in BMDMs after 3 days of 1 μM iBRD9 or vector treatment with or without RANKL-induction, as measured by qPCR. M-CSF, M; RANKL, R. n = 3 biologically independent samples. All data in this figure are represented as mean ± SD. Two-tailed Student’s t -test for ( a ) and ( b ). One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test for ( d ), ( e ), and ( f ); ANOVA with Tukey’s multiple comparisons test for ( h ). All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For osteoclast induction, BMDMs were cultured into complete media with 50 ng/ml recombinant soluble murine M-CSF (PeproTech, 315-02) and 100 ng/ml recombinant soluble murine RANKL (PeproTech, 315-11).

Techniques: Staining, Control, Expressing, Western Blot, Concentration Assay, Cell Counting, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a Heatmap hierarchical clustering and the MA plot showing the gene expression profiles in BMDMs after 1 day of BRD9 inhibition during osteoclastic differentiation with M-CSF and RANKL (MR). n = 4. Color scale represents normalized gene FPKM value by z-score scheme. b The top 10 Gene ontology (GO) biological process enriched in the comparison between BMDMs in MR + vector and MR + iBRD9 group . c Gene set enrichment analysis (GSEA) analysis of the top downregulated gene sets in MR + iBRD9 group compared with control group. d GSEA analysis of the top upregulated gene sets in MR + iBRD9 group compared with control group. e The protein expression of IFN-β, STAT2, and STAT1 in BMDMs at different days after osteoclastic differentiation, as measured by western blot. f GSEA plots and heatmap hierarchical clustering of GO term_0035458 in MR + iBRD9 group and control group. n = 4. g The mRNA expression of Acp5 and Mmp9 in BMDMs treated with 2 days of 1 μM iBRD9 and 0.0625 ng/ml IFN-β1 during osteoclastic induction, as measured by qPCR. n = 3 biologically independent samples. h TRAP staining of BMDMs treated with 3 days of 1 μM iBRD9 and 0.0625 ng/ml IFN-β1 during osteoclastic induction. Scale bar, 200 μm. All data in this figure are represented as mean ± SD. Hypergeometric distribution test for ( b ). Empirical phenotype-based permutation test for ( c ) and ( d ). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test for ( g ). All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For osteoclast induction, BMDMs were cultured into complete media with 50 ng/ml recombinant soluble murine M-CSF (PeproTech, 315-02) and 100 ng/ml recombinant soluble murine RANKL (PeproTech, 315-11).

Techniques: Gene Expression, Inhibition, Comparison, Plasmid Preparation, Control, Expressing, Western Blot, Staining

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a Venn diagram showing overlapping genes, where BRD9 binding, RANKL-induced while downregulated after BRD9 inhibition. b KEGG pathway annotation of the genes, where BRD9 binding, RANKL-induced while downregulated after BRD9 inhibition. c Protein-protein interaction (PPI) networks of the genes, where BRD9 binding, RANKL-induced while downregulated after BRD9 inhibition. d Immunofluorescence staining of STAT1 (red) and CTSK (green) in the mouse distal femur at 4 weeks of age. Scale bar,100 μm. e Immunofluorescence of STAT1 (green), TRAP staining (red) and actin (red) in BMDMs after 3 days of RANKL-induction. Scale bar, 100 μm. f The mRNA expression of Stat1 in BMDMs from LysM-Cre;Brd9 fl/fl mice compared with that from control littermates after 3 days of RANKL-induction. n = 3 for LysM-Cre;Brd9 fl/fl mice group. n = 6 for control littermates. g The mRNA expression of Stat1 in BMDMs treated with iBRD9 after 2 days of RANKL-induction. n = 3 biologically independent samples. h The protein expression of STAT1 in BMDMs treated with iBRD9 after 2 days of RANKL-induction. i The protein expression of STAT1 in RAW264.7 transfected with control lentivirus (NC) and Stat1 -overexpression lentivirus (OE-Stat1). j The mRNA expression of Mmp9 , Acp5 and Fos in Stat1 -overexpressed and control RAW264.7 treated with iBRD9 after 2 days of RANKL-induction. n = 4 biologically independent samples. k Luciferase reporter activities of the Stat1 promoter alone or in the presence of enhancer region in osteoclastic induced RAW264.7 treated with iBRD9 or control vector. n = 5 biologically independent samples. l ChIP assay with BRD9 antibody (or IgG) in RAW264.7 cell line during osteoclastic induction. n = 3 biologically independent samples. All data in this figure are represented as mean ± SD. Two-tailed Student’s t -test for ( f ), ( j ), ( k ), and ( l ). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test for ( g ). M, M-CSF; MR, M-CSF + RANKL. All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For osteoclast induction, BMDMs were cultured into complete media with 50 ng/ml recombinant soluble murine M-CSF (PeproTech, 315-02) and 100 ng/ml recombinant soluble murine RANKL (PeproTech, 315-11).

Techniques: Binding Assay, Inhibition, Immunofluorescence, Staining, Expressing, Control, Transfection, Over Expression, Luciferase, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a Heatmap showing the average ATAC-Seq signal centered on the transcription start site (TSS) of the nearest genes in osteoclastic induced BMDMs after control and iBRD9 treatment. Color scale represents average signal intensity. b Venn diagram showing the overlapping and discrepant peaks between control and iBRD9 group. c Volcano plot depicting differentially accessible region (DAR) between control and iBRD9 group. d GO terms of genes with gain and loss DAR around TSS after BRD9 inhibition. e Dot bubble plot showing the loss DAR motif enrichment after BRD9 inhibition. f Motifs enriched in BRD9 binding while loss after BRD9 inhibition. g The FOXP1 motif logo. h FOXP1 immunofluorescence (red) and CTSK (green) in the mouse distal femur at 4 weeks. Scale bar, 100 μm. i FOXP1 immunofluorescence (green), TRAP staining (red) and actin (red) in BMDMs after 3 days of RANKL-induction. Scale bar, 100 μm. j Co-IP assay with FOXP1 antibody (or IgG) in BMDMs during osteoclastic induction, followed by immunoblotting of BRD9 and FOXP1. k ChIP assay with FOXP1 antibody (or IgG) in RAW264.7 cell during RANKL-induction treated with iBRD9 or vector. n = 3 biologically independent samples. l The mRNA of Foxp1 , Stat1 , Myc , Mmp9 and Acp5 in osteoclastic induced RAW264.7 cell treated with Foxp1 knockdown lentivirus ( Foxp1 -Lenti) or control lentivirus (Control-Lenti). n = 3 biologically independent samples. m The mRNA of Foxp1 in BMDMs from LysM-Cre;Brd9 fl/fl ( n = 3) and control mice ( n = 6) at 4 weeks after 3 days of osteoclast differentiation. n The mRNA of Foxp1 in BMDMs treated with iBRD9 after 3 days of osteoclast differentiation. n = 3 biologically independent samples. o Schematic drawing shows chromatin remodeling mediated by BRD9. All data in this figure are represented as mean ± SD. Negative binomial distribution used for c . Hypergeometric distribution test for d . the one-tailed Fisher’s Exact test for ( e ) and ( f ). Two-tailed Student’s t -test for ( m ), ( n ), ( k ), and ( l ). MR, M-CSF + RANKL. All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For osteoclast induction, BMDMs were cultured into complete media with 50 ng/ml recombinant soluble murine M-CSF (PeproTech, 315-02) and 100 ng/ml recombinant soluble murine RANKL (PeproTech, 315-11).

Techniques: Control, Inhibition, Binding Assay, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Knockdown, One-tailed Test, Two Tailed Test

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a TRAP staining and quantification analysis of RANKL-induced BMDMs with 3 days of dBRD9 at different concentration. Scale bar, 200 μm. n = 7 biologically independent samples. b Cell viability of RANKL-induced BMDMs with 1 day of dBRD9 at different concentration, shown by cell counting kit 8 assay. n = 9 biologically independent samples. c The mRNA expression of Mmp9, Acp5, Dcstamp, and Stat1 in BMDMs after 2 days of 0.3 μM dBRD9 or vector treatment with or without RANKL-induction, as measured by qPCR. n = 6 biologically independent samples for Mmp9, Acp5, Dcstamp; n = 4 biologically independent samples for Stat1 . M-CSF, M; RANKL, R. d TRAP staining and quantification analysis of BMDMs treated with 3 days of 0.3 μM dBRD9 and 0.0625 ng/ml IFN-β1 during osteoclastic induction. Scale bar, 200 μm. n = 7 biologically independent samples. e The mRNA expression of Mmp9, Acp5 and Dcstamp in BMDMs treated with 2 days of 0.3 μM dBRD9 and 0.0625 ng/ml IFN-β1 during osteoclastic induction, as measured by qPCR. n = 3 biologically independent samples. M-CSF, M; RANKL, R. f Heatmap showing the change on the transcriptional profile of critical osteoclast-related genes between dBRD9 and JQ1 treated BMDMs during osteoclastogenesis. g GO analysis shows the enriched top ten changed signaling pathway in JQ1-treated group compared with the dBRD9-treated group. h Heatmap showing the cell cycle-related signature genes in JQ1-treated group compared with the dBRD9-treated group during osteoclastogenesis. All data in this figure are represented as mean ± SD. Hypergeometric distribution test for g . One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test for ( a ) and ( b ); with Tukey’s multiple comparisons test for ( c ), ( d ), and ( e ). Color scale in f and h represents normalized gene FPKM value by z-score scheme. All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For osteoclast induction, BMDMs were cultured into complete media with 50 ng/ml recombinant soluble murine M-CSF (PeproTech, 315-02) and 100 ng/ml recombinant soluble murine RANKL (PeproTech, 315-11).

Techniques: Staining, Concentration Assay, Cell Counting, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a Schematic illustrating the protocol used to establish ZOL-related ONJ model and dBRD9 degrader treatment. b μCT imaging of the maxillary bone from mice in dBRD9 degrader treated group and the control group. Yellow arrow 1 indicates severe periosteal reaction. Yellow arrow 2 indicates hyper-osseous maxillary sinus floor. Yellow arrow 3 indicates osteosclerotic patterns and sequestrum. Asterisk indicates insufficient amount of bone. Scale bar, 1 mm. c The incidence rate of osteonecrosis and periosteal reaction of the maxillary bone from mice in dBRD9 degrader treated group and the control group. n = 5. d H&E staining of the periodontal and alveolar bone area from mice in BRD9 degrader treated group and the control group. Colored boxes in the left are shown at higher magnification in right. Black dashed lines outline the boundary of the healed extraction sockets. Asterisk indicates necrotic alveolar bone with empty lacunae. Scale bar, 200 μm. e TRAP staining and f quantification of the periodontal and alveolar bone area from mice in BRD9 degrader treated group and the control group. Colored boxes in the left are shown at higher magnification in right. Arrows point to TRAP-positive osteoclasts. Asterisk indicates no signal. Scale bar, 200 μm. n = 5. Immunofluorescence staining of g iNOS (red), h TNF-a (red), i CD86 (red), k STAT1 (red) of the periodontal and alveolar bone area from mice in BRD9 degrader treated group and the control group. Colored boxes in the left are shown at higher magnification in right. Blue dashed lines outline the boundary of the healed extraction sockets. Arrows indicates positive signal. Scale bar, 200 μm. j The mRNA expression of Brd9 in ZOL or vector-treated BMDMs during LPS-induced macrophage activation and RANKL-induced osteoclastogenesis at indicated timepoint. n = 3 biologically independent samples. All data in this figure are represented as mean ± SD. Two-tailed Student’s t -test for ( f ) and ( j ). All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For osteoclast induction, BMDMs were cultured into complete media with 50 ng/ml recombinant soluble murine M-CSF (PeproTech, 315-02) and 100 ng/ml recombinant soluble murine RANKL (PeproTech, 315-11).

Techniques: Imaging, Control, Staining, Extraction, Immunofluorescence, Expressing, Plasmid Preparation, Activation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a Schematic illustrating the protocol for establishing LPS-induced localized aggressive periodontitis (LAP). b μCT imaging and d quantification of LPS-induced local alveolar bone loss in the maxillary bone from 4-week-old LysM-Cre;Brd9 fl/fl mice ( n = 4) and littermate control mice ( n = 9). Yellow line in b indicates cementum-enamel junction to alveolar bone crest (CEJ-ABC) distance (mm) in d . c H&E staining of LPS-stimulated maxillary bone from 4-week-old LysM-Cre;Brd9 fl/fl mice and littermate control mice. Arrows indicate distance between gingival sulcus bottom to alveolar bone crest. e TRAP staining and f quantification of LPS-stimulated maxillary bone from 4-week-old LysM-Cre;Brd9 fl/fl mice ( n = 3) and littermate control mice ( n = 4). g TNF-a, iNOS and RANKL immunofluorescence (green) in LPS-stimulated maxillary bone from 4-week-old LysM-Cre;Brd9 fl/fl mice and littermate control mice. h Schematic illustrating dBRD9 degrader treatment. i μCT imaging and j quantification of LPS-induced local alveolar bone loss in BRD9 degrader treated (right) and control (left) lateral of the maxillary bone from 6-week-old mice. n = 6. k H&E staining, l TRAP staining, and m quantification of LPS-induced local alveolar bone in BRD9 degrader treated and control lateral of the maxillary bone from 6-week-old mice. n = 6. n RANKL, TNF-a, and iNOS immunofluorescence (green) in LPS-induced local alveolar bone in BRD9 degrader treated and control lateral of the maxillary bone from 6-week-old mice. o The mRNA expression of TNF-a , iNOS , Il6 , Cd86, and Rankl of LPS-induced local alveolar bone in BRD9 degrader treated and control lateral of the maxillary bone from 6-week-old mice. n = 7. All data in this figure are represented as mean ± SD. Two-tailed Student’s t -test for ( d ), ( f ), ( j ), ( m ), and ( o ). M1, first molar. M2, second molar. Asterisk in b , c indicates less bone mass. Scale bar in b , i , 1 mm; c , e , k , l , 200 μm; g , n , 100 μm. All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For osteoclast induction, BMDMs were cultured into complete media with 50 ng/ml recombinant soluble murine M-CSF (PeproTech, 315-02) and 100 ng/ml recombinant soluble murine RANKL (PeproTech, 315-11).

Techniques: Imaging, Control, Staining, Immunofluorescence, Expressing, Two Tailed Test

Journal: Clinical and Developmental Immunology

Article Title: RANKL Cytokine: From Pioneer of the Osteoimmunology Era to Cure for a Rare Disease

doi: 10.1155/2013/412768

Figure Lengend Snippet: Preclinical studies in Rankl −/− mice. Rankl −/− mice were injected with either 1 mg/kg RANKL (below) or PBS (above) every 48 hours starting within the first week of life. Mice were sacrificed after 1 month (P30) or after 3 months (P90). Periodic acid-Schiff staining (bone in pink, cartilage in violet) of femur sections showed in RANKL-treated mice rescue of the bone defect at P30 and pathological reduction of the bone content at P90, clearly indicating overtreatment. Scale bar: 200 μ m.

Article Snippet: To test this hypothesis, we conducted a preclinical study treating Rankl −/− mice with subcutaneous injections of RANKL (soluble recombinant murine RANKL, amino acids 158–316, kindly provided by Amgen, Inc., Thousand Oaks, CA, USA; dosages 0.5, 1, or 2 mg/kg) from the first week of life, every 48 hours, for 1 month [ ].

Techniques: Injection, Staining