Journal: Nature Communications

Article Title: GDF11 decreases bone mass by stimulating osteoclastogenesis and inhibiting osteoblast differentiation

doi: 10.1038/ncomms12794

Figure Lengend Snippet: ( a ) Representative images of microCT reconstruction of distal femurs. Scale bar, 500 μm. ( b ) Von Kossa staining of undecalicified sections of femurs. Scale bar, 500 μm. ( c ) TRAP staining of femur sections from vehicle and rGDF11 treated mice. Scale bar, 50 μm. ( d ) Histomorphometric analysis of the metaphysis region of distal femurs. Results are shown as mean±s.d.; n =6–10; * P <0.05, ** P <0.01 and *** P <0.001 by analysis of variance (ANOVA) with Tukey's post hoc test. ( e , f ) The serum levels of CTX and P1NP. Results are shown as mean±s.d.; n =6–10; * P <0.05 by ANOVA with Tukey's post hoc test.

Article Snippet: For native condition, rGDF11 or Myostatin (#120-00, PeproTech, Rocky Hill, NJ) were added into the loading buffer (62.5 mM Tris-HCl, pH 6.8; 40% glycerol; and 0.01% Bromophenol Blue), and electrophoresed on 4–15% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA) in a running buffer containing 25 mM Tris Base and 192 mM glycine at 4 °C.

Techniques: Staining

Journal: Nature Communications

Article Title: GDF11 decreases bone mass by stimulating osteoclastogenesis and inhibiting osteoblast differentiation

doi: 10.1038/ncomms12794

Figure Lengend Snippet: ( a ) Representative images of microCT reconstruction of distal femurs. Scale bar, 500 μm. ( b ) Von Kossa staining of undecalicified sections of femurs. Scale bar, 500 μm. ( c ) TRAP staining of femur sections from vehicle and rGDF11 treated mice. Scale bar, 50 μm. ( d ) Histomorphometric analysis of the metaphysis region of distal femurs. Results are shown as mean±s.d.; n =6; * P <0.05 by t test. ( e ) Representative images of lumber 4 vertebrae stained with Von Kossa. Scale bar, 500 μm. ( f ) Histomorphometric analysis of the trabecular bone in vertebrae. Results are shown as mean±s.d.; n =6; * P <0.05 and ** P <0.01 by t test. ( g , h ) The serum levels of CTX and P1NP. Results are shown as mean±s.d.; n =6; ** P <0.01 by t test.

Article Snippet: For native condition, rGDF11 or Myostatin (#120-00, PeproTech, Rocky Hill, NJ) were added into the loading buffer (62.5 mM Tris-HCl, pH 6.8; 40% glycerol; and 0.01% Bromophenol Blue), and electrophoresed on 4–15% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA) in a running buffer containing 25 mM Tris Base and 192 mM glycine at 4 °C.

Techniques: Staining

Journal: Nature Communications

Article Title: GDF11 decreases bone mass by stimulating osteoclastogenesis and inhibiting osteoblast differentiation

doi: 10.1038/ncomms12794

Figure Lengend Snippet: ( a ) Representative TRAP staining of the BMMs after 4 days of osteoclast differentiation. 100 ng ml −1 M-CSF was present in all settings. Scale bar, 100 μm. ( b , c ) Osteoclast number and size. TRAP-positive cells with at least three nuclei were counted as osteoclasts. Results are shown as mean±s.d.; n =9; * P <0.05, ** P <0.01 and *** P <0.001 by analysis of variance (ANOVA) with Tukey's post hoc test. ( d , e ) Cell proliferation and apoptosis assay. BMMs were cultured in the presence of 100 ng ml −1 M-CSF and 50 ng ml −1 RANKL with indicated amount of rGDF11 for 2 days. Results are shown as mean±s.d.; n =8. ( f ) Representative images of resorption pits on Osseo Assay surface after 7 days of culture. Scale bar, 100 μm. ( g , h ) Resorption pits number and resorption area. Results are shown as mean±s.d.; n =8; ** P <0.01 and *** P <0.001 by ANOVA with Tukey's post hoc test. ( i ) Representative images of osteoclasts in co-cultures with osteoblasts in presence of 1,25(OH) 2 D 3 and PGE 2 with indicated amount of rGDF11 for 5 days. Scale bar, 100 μm. ( j , k ) Osteoclast number and size for the co-cultures. Results are shown as mean±s.d.; n =8. * P <0.05 and *** P <0.001 by ANOVA with Tukey's post hoc test.

Article Snippet: For native condition, rGDF11 or Myostatin (#120-00, PeproTech, Rocky Hill, NJ) were added into the loading buffer (62.5 mM Tris-HCl, pH 6.8; 40% glycerol; and 0.01% Bromophenol Blue), and electrophoresed on 4–15% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA) in a running buffer containing 25 mM Tris Base and 192 mM glycine at 4 °C.

Techniques: Staining, Apoptosis Assay, Cell Culture

Journal: Nature Communications

Article Title: GDF11 decreases bone mass by stimulating osteoclastogenesis and inhibiting osteoblast differentiation

doi: 10.1038/ncomms12794

Figure Lengend Snippet: ( a ) Gene expression levels of the BMMs stimulated without or with 100 ng ml −1 rGDF11 for 24 h (three biological replicates per group). 467 genes (red) were upregulated and 679 genes (black) were downregulated. 100 ng ml −1 M-CSF was present in all settings. ( b ) Heatmap of the osteoclastogenesis associated genes. ( c ) Quantitative RT-PCR confirmed the increased expression of osteoclast key marker genes. Results are shown as mean±s.d.; n =3. *** P <0.001 by t test. ( d ) KEGG pathway analysis indicated the altered function of TGF-β pathway. ( e ) Heatmap of the TGF-β pathway associated genes. ( f ) Western blot analysis indicated that rGDF11 stimulated the phosphorylation of Smad2/3 in BMMs in 30 min. ( g ) Western blot analysis demonstrated that rGDF11 amplified the RANKL-induced expression of c-Fos. BMMs were starved overnight and then treated for 4 h. ( h ) Representative images of immunohistochemical staining. rGDF11 injections increased the phosphorylation of Smad2/3 and c-Fos, as well as the expression of Nfatc1 in vivo . Femurs were collected ∼2 h after the last injection of rGDF11. Scale bar, 50 μm. ( i ) Western blot analysis indicated that rGDF11 stimulated the RANKL-induced expression of Nfatc1. BMMs were treated for 2 days. ( j ) ChIP assay revealed that rGDF11 induced the co-occupancy of Smad2/3 and c-Fos to the binding region of Nfatc1 . Results are shown as mean±s.d.; n =3. * P <0.05 and ** P <0.01 by t test. ( k ) Western blot analysis of Nfatc1. Depletion of c-Fos eliminated the rGDF11 induced expression of Nfatc1. ( l ) ChIP assay. Depletion of c-Fos abolished rGDF11 triggered binding of Smad2/3 to Nfatc1 . Results are shown as mean±s.d.; n =3. ** P <0.01 by t test.

Article Snippet: For native condition, rGDF11 or Myostatin (#120-00, PeproTech, Rocky Hill, NJ) were added into the loading buffer (62.5 mM Tris-HCl, pH 6.8; 40% glycerol; and 0.01% Bromophenol Blue), and electrophoresed on 4–15% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA) in a running buffer containing 25 mM Tris Base and 192 mM glycine at 4 °C.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Marker, Western Blot, Phospho-proteomics, Amplification, Immunohistochemical staining, Staining, In Vivo, Injection, Binding Assay

Journal: Nature Communications

Article Title: GDF11 decreases bone mass by stimulating osteoclastogenesis and inhibiting osteoblast differentiation

doi: 10.1038/ncomms12794

Figure Lengend Snippet: ( a ) Representative images of ALP staining and Alizarin Red S (ARS) staining of BMSCs. ( b , c ) Quantitative analyses of the ALP activity and calcium mineralization in BMSCs. Results are shown as mean±s.d.; n =5; * P <0.05 and ** P <0.01 by t test. ( d ) Quantitative RT-PCR revealed reduced messenger RNA expression of Runx2 , Osx , Alp and Ocn in rGDF11 treated BMSCs. Results are shown as mean±s.d.; n =5; * P <0.05, ** P <0.01 and *** P <0.001 by t test. ( e ) Representative images of ALP and ARS staining of primary calvarial osteoblasts. ( f , g ) Quantitative analyses of the ALP activity and calcium mineralization in osteoblasts. Results are shown as mean±s.d.; n =5; *** P <0.001 by t test. ( h ) Quantitative RT-PCR demonstrated that rGDF11 inhibited messenger RNA expression of Runx2 , Osx , Alp and Ocn in osteoblasts. n =3. Results are shown as mean±s.d.; n =5; ** P <0.01 and *** P <0.001 by t test. ( i ) Western blot indicated that rGDF11 stimulated the phosphorylation of Smad2/3 in osteoblasts. ( j ) ChIP assay revealed that rGDF11 reduced the abundance of acetylated histone H4 (H4ac) on the promoter of Ocn . Results are shown as mean±s.d.; n =3; ** P <0.01 by t test. ( k , l ) Western blot analysis of pSmad1/5. The presence of rGDF11 attenuated BMP2 or foetal bovine serum induced phosphorylation of Smad1/5. Osteoblasts were starved overnight and then treated for 30 min.

Article Snippet: For native condition, rGDF11 or Myostatin (#120-00, PeproTech, Rocky Hill, NJ) were added into the loading buffer (62.5 mM Tris-HCl, pH 6.8; 40% glycerol; and 0.01% Bromophenol Blue), and electrophoresed on 4–15% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA) in a running buffer containing 25 mM Tris Base and 192 mM glycine at 4 °C.

Techniques: Staining, Activity Assay, Quantitative RT-PCR, RNA Expression, Western Blot, Phospho-proteomics

Journal: Nature Communications

Article Title: GDF11 decreases bone mass by stimulating osteoclastogenesis and inhibiting osteoblast differentiation

doi: 10.1038/ncomms12794

Figure Lengend Snippet: ( a ) Representative images of microCT reconstruction of femoral cortical bone defects. The red dotted lines indicate the position of the original defect margin. Scale bar, 500 μm. ( b ) H&E staining of femoral cortical bone defects. The black dotted lines indicate the position of the original defect margin. Abbreviation: CB, cortical bone. Scale bar, 200 μm. ( c ) Bone mineral density (BMD) and histomorphometric analysis of the regenerated bone in femoral cortical gaps. Results are shown as mean±s.d. The mean values of the bilateral defects of each mouse were counted as individual data points ( n =5). * P <0.05, ** P <0.01 and *** P <0.001 by t test. ( d ) Representative images of TRAP staining of the regenerated bone in femoral cortical gaps. Scale bar, 50 μm. ( e ) Representative images and quantification of immunohistochemical staining of pSmad2/3. rGDF11 injections increased the phosphorylation of Smad2/3. Scale bar, 50 μm. ( f ) MicroCT reconstruction of the calvarial defects. The red dotted lines indicate the position of the original defect margin. Scale bar, 1 mm. ( g ) H&E staining of the calvarial defects. The dotted lines indicate the position of the original defect margin. CB, cortical bone. Scale bar, 200 μm. ( h ) Bone mineral density (BMD) and histomorphometric analysis of the regenerated bone in calvarial defects. Results are shown as mean±s.d. The mean values of the bilateral defects of each mouse were counted as individual data points ( n =5). * P <0.05 and ** P <0.01 by t test.

Article Snippet: For native condition, rGDF11 or Myostatin (#120-00, PeproTech, Rocky Hill, NJ) were added into the loading buffer (62.5 mM Tris-HCl, pH 6.8; 40% glycerol; and 0.01% Bromophenol Blue), and electrophoresed on 4–15% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA) in a running buffer containing 25 mM Tris Base and 192 mM glycine at 4 °C.

Techniques: Staining, Immunohistochemical staining, Phospho-proteomics