Journal: Science signaling

Article Title: Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice

doi: 10.1126/scisignal.abd0536

Figure Lengend Snippet: (A) Images of μCT scans of ectopic mineralized tissue masses present in mice implanted with Matrigel-rhBMP2 mixtures and then treated twice per week with control pre-immune IgGb2 mouse monoclonal antibody (BMP2 + IgG) or neutralizing activin A mouse monoclonal IgGb2 antibody (BMP2 + nActA.Ab). Samples were harvested and processed for analyses on day 14 after Matrigel implantation. Images show mineralized masses from 4 different mice in each treatment group. (B) Images of μCT scans of ectopic masses on day 14 in mice implanted with Matrigel-rhBMP6 mixtures and administered pre-immune or neutralizing activin A antibody as in (A). (C and D) Quantification of bone volume relative to total volume (BV/TV) in ectopic masses retrieved from the mice implanted with Matrigel-rhBMP2 (C) and Matrigel-BMP6 (D) and treated with pre-immune or neutralizing activin A antibody. Data are presented a mean ± S.D. N = 6–10 ectopic masses per experimental group, 2 masses per mouse, obtained from 3 independent experiments. Student’s t tests were used in all experiments to determine statistical significance. **P<0.01; ****P<0.0001. Scale bar for all panels, 1 mm.

Article Snippet: Thus, we treated micromass cultures with rhBMP6 in the absence and presence of SB431542.

Techniques: Control

Journal: Science signaling

Article Title: Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice

doi: 10.1126/scisignal.abd0536

Figure Lengend Snippet: (A) Images and quantification of alcian blue staining of E11.5 limb bud mesenchymal cells in micromass cultures treated with recombinant activin A (100 ng/ml) or rhTGF-β1 (5 ng/ml) in the absence (−) or presence (+) of the pSMAD2/3 signaling antagonist SB431542 (10 μM), starting about 2 hrs after plating. Control cultures received vehicle only. Alcian blue staining was quantified at day 3 using ImageJ and Particle Analysis software. (B) Expression of chondrogenic genes Sox9, Acan, and Col2a1 in day 3 control versus activin A–treated cultures in (A). (C) Immunoblot analysis and quantification of phosphorylated SMAD2 (pSMAD2) and SMAD2/3 in limb bud mesenchymal cell cultures that were acutely treated for 1 hr with activin A, TGF-β1, and the pSMAD2/3 inhibitor SB431542 as indicated. Molecular weight markers are in the left-most lane. Protein loading consistency was assessed by re-processing the blots with antibodies specific for nonphosphorylated SMAD2/3 and GAPDH. (D) Images and quantification of alcian blue staining of micromass cultures treated with rhBMP6, SB431542, or both and processed on day 3. (E) RT-PCR quantification of Sox9, Acan, and Col2a1 expression in day 3 control versus treated and co-treated cultures in (D). (F) Immunoblot analysis and quantification of pSMAD1/5/8 in micromass cultures after 1 hr treatment with rhBMP6 and SB431542 as indicated. Molecular weight markers are in the left-most lane. Loading was assessed by re-blotting for SMAD1 and GAPDH. (G) Images and quantification of alcian blue staining in micromass cultures on day 3 treated with rhBMP6 (50 ng/ml) alone or in combination with activin A (100 ng/ml) starting about 2 hrs from plating. (H) RT-PCR quantification of Sox9, Acan, and Col2a1 expression in day 3 control versus BMP6-treated and BMP6 and activin A co-treated cultures. (I) Immunoblot analysis and quantification of pSMAD1/5/8 in micromass cultures treated for 1 hr with rhBMP6 and activin A as indicated. Molecular weight markers are in the left-most lane. Loading was assessed by re-blotting for SMAD1 and GAPDH. Statistical significance was determined by Student’s t tests and one-way ANOVA in all experiments. *P<0.05; **P<0.01; ****P<0.0001; #P<0.05; ##P<0.01; ####P<0.0001; $ $<0.01. * indicates a comparison to the control; # indicates a comparison to BMP6 or Activin A; and $ indicates a comparison to TGF-β1. N = 3 to 5 independent experiments with 3 repeats per sample, and data are presented as mean ± S.D.

Article Snippet: Thus, we treated micromass cultures with rhBMP6 in the absence and presence of SB431542.

Techniques: Staining, Recombinant, Control, Particle Size Analysis, Software, Expressing, Western Blot, Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: Science signaling

Article Title: Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice

doi: 10.1126/scisignal.abd0536

Figure Lengend Snippet: (A and B) μCT images of intramuscular mineralized tissue masses in mice implanted with Matrigel-rhBMP2 (A) or Matrigel-rhBMP6 (B) mixtures and then treated twice per week with control pre-immune mouse monoclonal IgG (IgG) or neutralizing activin A mouse monoclonal antibody (nActA.Ab) as indicated. Samples were harvested and processed for analyses on day 14 from implantation. (C and D) Quantification of bone volume (BV) within the ectopic masses retrieved from the indicated mouse groups. Data are from indicated numbers of mice (2 data point per mouse) from a minimum of 3 independent experiments and are plotted as mean ± S.D. N = 6–10 ectopic masses, 2 masses per mouse, per experimental group. Student’s t tests were used in all experiments to determine statistical significance. *P<0.05; **P<0.01. Scale bar, 1 mm

Article Snippet: Thus, we treated micromass cultures with rhBMP6 in the absence and presence of SB431542.

Techniques: Control