C2C12 and murine mesenchymal cells (C3H10T1/2) were cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% FBS and antibiotics. The cells were passaged before reaching confluence and used within 10 passages. To induce tenocyte differentiation, C2C12 cells were cultured in serum‐free AIM‐V medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with GDFs (500 ng·mL−1 of GDF‐5, ‐6, ‐7 and 10, 100, 500 ng·mL−1 of myostatin; R&D Systems, Minneapolis, MN, USA). The medium was replaced every 2 days. To analyze signaling pathways, cells were treated with 500 ng·mL−1 of myostatin and 10 μmol·L−1 of inhibitors for ALK (SB431542), p38MAPK (SB203580), and MEK1 (PD98059) (Abcam, Cambridge, MA, USA) for 5 days.
Myostatin
Manufactured by Abcam
Sourced in United States, United Kingdom
Myostatin is a protein that plays a key role in regulating muscle growth and development. It is a member of the transforming growth factor-beta (TGF-β) superfamily of proteins. Myostatin acts as a negative regulator of muscle mass, limiting the growth and size of muscle fibers.
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Lab products found in correlation
Fitc anti mouse cd11b, by BioLegend (2 mentions)
Myostatin, by R&D Systems (1 mentions)
Sb431542, by Abcam (1 mentions)
Gdf 5, by R&D Systems (1 mentions)
Sb203580, by Abcam (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Pd98059, by Abcam (1 mentions)
Anti tgf β, by Abcam (1 mentions)
Murf1, by Santa Cruz Biotechnology (1 mentions)
Tgf β, by Abcam (1 mentions)
Anti p smad2 3, by Santa Cruz Biotechnology (1 mentions)
P smad2 3, by Cell Signaling Technology (1 mentions)
Smad2, by Cell Signaling Technology (1 mentions)
Anti p21, by Santa Cruz Biotechnology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Gs 800, by Bio-Rad (1 mentions)
10 protocols using myostatin
1
Tenocyte Differentiation of C2C12 and C3H10T1/2 Cells
2
Western Blot Analysis of Muscle Atrophy
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For Western blotting, total protein was isolated from mouse quadriceps or adipose tissue by homogenization in RIPA buffer with complete protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche, Pleasanton, CA)[33 (link)]. Protein quantification was determined by the Bradford protein assay, and samples were equally loaded on 10% polyacrylamide gels (NuPage, Grand Island, NY), electrophoresed at 200V, electrotransferred to PVDF membranes, and probed with antibodies to Atrogin-1 (1:500; ECM Biosciences, Versailles, KY), Myostatin (1:200; Abcam, Cambridge, MA), MuRF1, ZAG (1:500; both Santa Cruz Biotechnology, Dallas, TX), p21 (1:200; Santa Cruz), β-actin, Smad2, p-Smad2/3 (1:1000; all Cell Signaling, Danvers, MA), TGF-β (1:1000; Abcam), and Ezrin (1:1000; BD Transduction Laboratories, San Jose, CA). Quantification was performed by measuring integrated density and normalizing to loading controls. For immunohistochemical analysis, paraffin-embedded tissue sections were stained using polyclonal anti-TGF-β (1:100; Abcam, Cambridge, MA), anti-p21 (1:100, Santa Cruz), or anti p-Smad 2/3 (1:50, Santa Cruz) and the corresponding isotype controls[33 (link)].
3
Protein Analysis of Primary Tissues
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Total proteins from primary tissues and cell lines were extracted in lysis buffer (Thermo Fisher Scientific, Rockford, IL, United States) and quantified using the Bradford method. Fifty micrograms of protein were separated by 12% SDS–PAGE. After transferring, the polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States) were incubated overnight at 4 °C with the following antibodies TAZ (560235, 1:1000; Becton Dickinson, Franklin Lakes, NJ, United States), myostatin (98337, 1:1000; Abcam, Cambridge, MA, United States), Akt (#9272, 1:500; GST), p-Akt (4060S, 1:500; GST), forkhead box (FOX)O4 (#9472, 1:500; GST), and GAPDH (TA-08, 1:500; ZSGB-BIO, Beijing, China). After incubation with peroxidase-coupled anti-mouse IgG (Beyotime, Jiangsu, China) at 37 °C for 2 h, bound proteins were visualized using Bio-Rad GS-800 and analyzed using Image J software. The relative protein levels were calculated based on GAPDH as the loading control.
4
Skeletal Muscle Protein and Gene Expression Analysis
5
Antibodies and PCR Primers Sourcing
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Antibodies for myostatin and MMP‐9 were purchased from Abcam (Cambridge, MA), EMMPRIN and iNOS antibodies and all secondary antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). The glyceraldehyde 3‐phosphate dehydrogenase (GAPDH ) antibody was purchased from EMD Millipore (Billerica, MA). All PCR primers were purchased from Invitrogen (Carlsbad, CA).
6
Muscle Biopsy Analysis of BMD
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An open muscle biopsy was performed on the nondominant biceps brachii under local anesthesia with separate quadrants sampled at baseline and 8 weeks in six patients. The biceps brachii was chosen due to relative preservation of this muscle in BMD vs shoulder girdle, pelvic girdle, and thigh musculature and the lack of deleterious effect of biopsy on lower extremity clinical testing. The western blot methodology is detailed in the Supporting Information online. All specimens were assessed with the technician blinded to the baseline vs treatment status. Antibodies used for quantification of bioenergetic signaling proteins, mitochondrial proteins, structural muscle proteins, indicators of regeneration, and regulators of muscle growth were from the following sources: 5′‐adenosine monophosphate (AMP)‐activated protein kinase (AMPK), LKB1, PGC1α, mitofilin dysferlin, follistatin, myostatin, Myf5, MyoD, myogenin, and MEF2a (all from Abcam, Cambridge, Massachusetts, USA); dystrophin, utrophin, creatine kinase, myosin, and actin α1 (all from Santa Cruz Biotechnology, Santa Cruz, California); and glyceraldehyde 3‐phosphate dehydrogenase (all from Cell Signaling, Danvers, Massachusetts), which was used as loading control. Band intensities were digitally quantified using ImageJ software (National Institutes of Health, Bethesda, Maryland; http://www.nih.gov ).
7
Modulation of Muscle Macrophage Signaling
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The HES used in this study was obtained from Tokyo Chemical Industry (Tokyo, Japan), dissolved in distilled water, and administered orally at a dose of 5 or 10 mg/kg/day daily to mice.
For the flow cytometry analysis, FITC anti-mouse CD11b, AF647 anti-mouse F4/80, PE anti-mouse CD163, PerCP/Cyanine5.5 anti-mouse CD206, FITC anti-mouse CD45, and PE anti-mouse CD86 antibodies were purchased from BioLegend (San Diego, CA, USA). Specific antibodies against phospho-forkhead box O3a (p-FoxO3a), FoxO3a, phospho-AKT (p-AKT), AKT, phospho-mTOR (p-mTOR), mTOR, phospho-p70S6 kinase (p-p70S6K), and p70S6K were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Fbx32, MuRF1, myostatin, and MyoD were purchased from Abcam (Cambridge, UK). Antibodies against phosphoinositide 3-kinase (PI3K), myogenin, and myocyte enhancer factor 2 (MEF-2) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
For the flow cytometry analysis, FITC anti-mouse CD11b, AF647 anti-mouse F4/80, PE anti-mouse CD163, PerCP/Cyanine5.5 anti-mouse CD206, FITC anti-mouse CD45, and PE anti-mouse CD86 antibodies were purchased from BioLegend (San Diego, CA, USA). Specific antibodies against phospho-forkhead box O3a (p-FoxO3a), FoxO3a, phospho-AKT (p-AKT), AKT, phospho-mTOR (p-mTOR), mTOR, phospho-p70S6 kinase (p-p70S6K), and p70S6K were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Fbx32, MuRF1, myostatin, and MyoD were purchased from Abcam (Cambridge, UK). Antibodies against phosphoinositide 3-kinase (PI3K), myogenin, and myocyte enhancer factor 2 (MEF-2) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
8
Muscle Protein Expression Analysis
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Antibodies against myoblast determination protein 1 (MyoD), F-box protein (Fbx32, also known as atrogin), muscle ring-finger 1 (MuRF1), and myostatin were bought from Abcam (Cambridge, UK). Antibodies against myogenin, myocyte enhancer factor 2 (MEF-2), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPx1), catalase, and α-tubulin were bought from Santa Cruz Biotechnology (Dallas, TX, USA). FITC anti-mouse CD11b and APC anti-mouse F4/80 were bought from BioLegend (San Diego, CA, USA).
9
Protein Extraction and Analysis from Tumors
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Proteins from tumors were extracted with a buffer solution (20 mM Tris-HCl, pH 7.5, 2 mM ATP, 5 mM MgCl2, 1 mM dithiothreitol (DTT), and 5 μL of a protease inhibitor cocktail (Sigma)). Proteins (20 μg/lane) were separated on a 12.5% polyacrylamide gel (a precast SDS gel (Bio-Rad)) and then transferred to a polyvinylidene difluoride membrane (Immobilon, Millipore). Proteins were determined using antibodies against mouse IL-6 (1:200, Abcam), TNF (1:200, Santa Cruz Biotechnology), Myostatin (1:100, Abcam), MAFbX (1:200, Santa Cruz Biotechnology), MuRF 1 (1:100, Novus), Cathepsina L (1:100, Abcam), Calpain (1:100, Santa Cruz Biotechnology), p-IκB-α (1:100, Santa Cruz Biotechnology) and FOXO1 (1:200, Abcam). The antibodies then were stripped off the membrane and re-probed with a specific antibody against β-actin (1:5000, Novus Biologicals). The intensity was quantified using the Fotodyne Image analysis System (Fotodyne, Hartland, WI, USA) and the TotalLab software (Nonlinear Dynamics, Durham, NC, USA).
10
Quantitative Analysis of Vascular Remodeling
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Formaldehyde fixed cuffed femoral arteries were paraffin-embedded and 5µm thick cross sections of arteries were stained to visualize vessel morphology. For quantification of intimal thickening, elastic laminae were visualized with Weigert’s elastin staining. For immunohistochemical analysis, the following antibodies were used: Myostatin (ab71808; Abcam, Cambridge, UK), BrdU (ab221240; Abcam), Ki-67 (ab16667; Abcam), αSMA (1A4 clone, Dako), Mac-3 (550292; BD Pharmingen). Images of stained slides were obtained using Panoramic 250 Flash III (3DHISTECH). All quantifications were performed on six sequential representative sections per vessel segment using image analysis software (Qwin, Leica) for Weigert’s elastin staining. All other quantifications were done using Panoramic Viewer software (3DHISTECH).
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