Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C is a positive regulator of human osteoclastogenesis. a–e Human osteoclast precursor cells were nucleofected with control or two different MEF2C siRNAs. a, b MEF2C mRNA and protein expression. c, d Osteoclastogenesis assay. c TRAP staining of human osteoclasts. Scale bar, 100 μm. d Osteoclast number of three independent experiments. e RT-qPCR analysis of ITGB3 , CTSK , and CTR mRNA after 72 h of culture with or without RANKL (40 ng·mL −1 ) normalized relative to TBP mRNA. Control samples without RANKL were set at 1.0. ( n = 3). f–h Human osteoclast precursor cells were transduced with adenoviral particles encoding GFP or MEF2C-FLAG. f TRAP staining of human osteoclasts transduced with GFP or MEF2C. Scale bar, 100 μm. g Cumulative data showing numbers of osteoclasts from six independent experiments. h Representative images of bone resorption assay of human osteoclasts transduced with GFP or MEF2C. i Quantitation of resorption area from six different experiments. Statistics used: a , d repeated measurement one-way ANOVA e repeated measurement Two-way ANOVA, g , i paired t -test. * P < 0.05, ** P < 0.01, *** P < 0.001. Data were shown as mean ± SD

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Control, Expressing, Staining, Quantitative RT-PCR, Transduction, Quantitation Assay

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C ΔMX mice show increased bone mass with decreased osteoclast numbers. a μCT analysis of femurs from 16-week-old male MEF2C ΔMX KO ( n = 7) and littermate control WT mice ( n = 9). Right panels show the indicated parameters in distal femurs. Bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular numbers (Tb.N), and porosity were determined by μCT analysis. b Histomorphometry analysis of the distal femur of 16-week-old male mice. Representative images showing TRAP-positive, multinucleated osteoclasts (red). Scale bars, 500 μm. Right panels show number of osteoclasts per bone surface (N.Oc/BS), osteoclast surface area per bone surface (Oc.S/BS) and eroded surface per bone surface (ES/BS). c Dynamic bone histomorphometry analysis of the distal femur. Scale bars, 50 μm. Representative images showing casein incorporation into newly calcifying bone. Right panels showed mineral apposition rate (MAR) and bone formation rate (BFR/BS). Data are shown as mean ± SD. Statistics used: a, b, c Welch’s t -test. NS; not significant, * P < 0.05, ** P < 0.01

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Control

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C-deficient cells show impaired osteoclastogenesis. a Representative image of TRAP staining of mouse osteoclasts. Scale bar, 100 μm. b Cumulative data showing numbers of osteoclasts from 6 independent experiments. c RT-qPCR analysis of Itgb3 , Ctsk and Ctr mRNA after 72 h of culture with or without RANKL (50 ng·mL −1 ) normalized relative to Hprt mRNA. Control samples without RANKL were set at 1.0. WT; n = 7, KO; n = 8. Data are shown as mean ± SD. Statistics used: b Welch’s t -test, c Two-way ANOVA. ** P < 0.01, *** P < 0.001

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Staining, Quantitative RT-PCR, Control

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C regulates expression of FOS and c-FOS-target genes in RANKL-stimulated human OCPs. a Heatmap showing relative expression (z-score) of 202 genes differentially expressed with P < 0.01 in RANKL-stimulated MEF2C KD cells versus RANKL-stimulated control cells from three biological replicates. b Enriched transcription factor binding motifs in region ±2 kb relative to transcription start site in RANKL-regulated MEF2C-dependent genes, by gene set enrichment analysis (GSEA). c Volcano plot of RNA-seq analysis of differentially expressed genes in human macrophages transduced with control or MEF2C#2 siRNAs. MEF2C and eight other genes obtained from b are marked. Of note, LEF1 and FOXF2 were filtered out due to low expression level (CPM < 3). Red dots, genes with q value < 0.05 (34 genes). d Cumulative values for FOS in RANKL-stimulated samples from RNA-sequencing with three biological replicates. Data are shown as mean ± SD

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Expressing, Control, Binding Assay, RNA Sequencing, Transduction

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: c-FOS mRNA and protein expression are dependent on MEF2C in human and mouse OCPs. Human osteoclast precursor cells were nucleofected with control or MEF2C #2 siRNAs. a RT-qPCR analysis of human FOS mRNA after 24 h of culture with or without RANKL normalized relative to TBP mRNA (control samples without RANKL set at 1.0). n = 5. b Representative immunoblotting of c-FOS in nuclear lysates. Lamin B1 and α-tubulin were used as controls for nuclear and cytoplasmic proteins, respectively. c Densitometric quantitation of c-FOS band intensity after 24 h of culture with RANKL from three independent donors. Mouse osteoclast precursor cells from MEF2C ΔMX KO or littermate control WT mice were cultured with M-CSF and RANKL. d RT-qPCR analysis of mouse Fos mRNA after 24 h of culture with or without RANKL (50 ng·mL −1 ) normalized relative to Hprt mRNA (control samples without RANKL set at 1.0). WT; n = 11, KO; n = 12. e Representative images of immunoblotting analysis of c-FOS expression in nuclear lysates. Right panel, densitometric quantitation of band intensity from four samples of each genotype. f Densitometric quantitation of c-FOS band intensity ( n = 4). Human osteoclast precursor cells were transduced with adenoviral particles encoding GFP or MEF2C-FLAG and stimulated with RANKL. g RT-qPCR analysis of human Fos mRNA after 12 h of culture with or without RANKL (40 ng·mL −1 ) normalized relative to TBP mRNA (control samples without RANKL set at 1.0). n = 8. h Immunoblotting of c-FOS at the indicated times. Representative images from five independent experiments. i Densitometric quantitation of c-FOS band intensity from five independent experiments. Data are shown as mean ± SD. Statistics used: a, d, g, i repeated measurement two-way ANOVA, c, f paired t -test. * P < 0.05, ** P < 0.01

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Quantitation Assay, Cell Culture, Transduction

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C binds to the upstream region of FOS gene and regulates NFATC1 expression. a A schematic view of FOS upstream regions with putative MEF2C binding sites predicted based on the analysis of MEF2C ChIP-sequencing data. b ChIP-qPCR analysis of the three putative MEF2C binding sites in the FOS upstream region. MEF2C binding was assessed in human OCPs transduced with adenoviral particles encoding GFP or MEF2C-FLAG and immunoprecipitated with FLAG antibodies. Dotted line represents enrichment level in the negative control HBB region in GFP transduced samples. ( n = 4) Mouse osteoclast precursor cells from MEF2C ΔMX KO or littermate control WT mice were transduced with retroviral particles encoding GFP or FOS and cultured with M-CSF and RANKL. c Representative image of TRAP staining of mouse osteoclasts. Scale bar, 100 μm. d Cumulative data showing numbers of osteoclasts from four independent experiments. Human osteoclast precursor cells were nucleofected with control or MEF2C #2 siRNAs. e RT-qPCR analysis of human NFATC1 mRNA after 48 h of culture with or without RANKL normalized relative to TBP mRNA (control samples with RANKL set at 1.0). n = 5. f Representative immunoblot of human NFATc1. p38 was used as a loading control. Right panel, densitometric quantitation of band intensity from three donors. Mouse osteoclast precursor cells from MEF2C ΔMX mice or littermate control WT mice were cultured with M-CSF and RANKL. g RT-qPCR analysis of mouse Nfatc1 mRNA after 24 h of culture with or without RANKL (50 ng·mL −1 ) normalized relative to Hprt mRNA (control samples without RANKL set at 1.0). WT; n = 11, KO; n = 12. h Representative images of immunoblotting analysis of mouse NFATc1 expression. Right panel, densitometric quantitation of band intensity from 4 samples of each genotype. Data are shown as mean ± SD. Statistics used: e repeated measurement two-way ANOVA, f paired t -test, g two-way ANOVA, h Welch’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Expressing, Binding Assay, ChIP-sequencing, ChIP-qPCR, Transduction, Immunoprecipitation, Negative Control, Control, Retroviral, Cell Culture, Staining, Quantitative RT-PCR, Western Blot, Quantitation Assay

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C deficient mice show attenuated pathological bone resorption in the K/BxN serum transfer arthritis model. a Schematic of experiments. b Time course of joint swelling and clinical score of K/BxN serum transfer arthritis in WT and MEF2C ΔMX KO mice. WT; n = 5, KO; n = 6. c TRAP staining of histological sections of hind paw and histomorphometric analysis of tarsal bones. Arrow, osteoclasts. Scale bars, 400 μm. Data are shown as mean ± SD. Statistics used: c Welch’s t -test * P < 0.05

Article Snippet: Anti-MEF2C antibody (Aviva Systems Biology #OAGA02537, San Diego, USA), Rabbit IgG (Control Antibody) (Vector Laboratories #I-1000, Burlingame, CA,USA), Citrate Buffer pH 6.0 (Sigma-Aldrich #C9999-1000ML), VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG) (Vector Laboratories), and DAB (Vector Laboratories #SK-4100) were used for immunohistochemistry.

Techniques: Staining