Journal: Journal of cachexia, sarcopenia and muscle

Article Title: A novel splice variant of the human MSTN gene encodes a myostatin-specific myostatin inhibitor.

doi: 10.1002/jcsm.13314

Figure Lengend Snippet: Figure 5 Myostatin-specific inhibition by myostatin-b. HeLa cells cotransfected with Luc and Gal plasmids were treated with recombinant myostatin at concentrations of 0, 20, 40 and 60 ng/mL (A); recombinant growth differentiation factor 11 (GDF11) at concentrations of 0, 10, 20 and 30 ng/mL (B); recombinant transforming growth factor β1 (TGF-β1) at concentrations of 0, 0.5, 1 and 2 ng/mL (C); and activin A at concentrations of 0, 10, 20 and 30 ng/mL (D). In addition, the myostatin-b plasmid was cotransfected into each treated cell. The results of luciferase activity are shown. Myostatin-b inhibited myostatin signalling induced only by recombinant myostatin but not by recombinant GDF11, recombinant TGF-β1 or recombinant activin A (red bars). The data are expressed as the mean ± SEM of three independent experiments. **P < 0.01, ***P < 0.001. RLU, relative luminescence unit.

Article Snippet: After 24 h, the medium was replaced with serum-free RPMI 1640 or DMEM containing recombinant myostatin (788-G8, R&D Systems), recombinant growth differentiation factor 11 (GDF11) (1958-GD, R&D Systems), recombinant TGF-β1 (240-B/CF, R&D Systems) or recombinant activin A (338-AC/CF, R&D Systems).

Techniques: Inhibition, Recombinant, Plasmid Preparation, Luciferase, Activity Assay

Journal: Skeletal Muscle

Article Title: Growth differentiation factor 11 induces skeletal muscle atrophy via a STAT3-dependent mechanism in pulmonary arterial hypertension

doi: 10.1186/s13395-022-00292-x

Figure Lengend Snippet: MCT-induced PAH caused body weight loss and muscle atrophy. A Circulating GDF11 levels in PAH patients and health control ( n =8). B RVSP of rats 4 weeks after MCT-treated, C body weight, and D the weight of gastrocnemius muscle, soleus muscle, tibialis anterior, and Extensor Digitorum Longus normalized per body weight (BW). E The weight of gastrocnemius muscle. F Representative images of EDL, TA, Sol, GM, and the cross-sectional areas of approximately 250 myofibers per group were determined. Scale bar represents 25 μm. G The distribution of myofiber cross-sectional area. n = 8 rats/group. Data are shown as mean ± SEM. Versus vehicle control, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Antibodies against p-STAT3 (Tyr705) (#9145), STAT3 (#9139), p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828), and Smad2/3 (#8685) were purchased from Cell Signaling Technology (Beverly, MA); antibodies against GDF11 (sc-81952) and SOCS3 (sc-51699) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against Fbx32 (ab168372), CD31 (ab28364), and wheat germ agglutinin (ab178444) were purchased from Abcam (Cambridge, MA); recombinant GDF11 protein (1958-GD) were purchased from R&D Systems (Minneapolis, MN); antibodies against Trim63 (55456-1-AP), ubiquitin (10201-2-AP), iNOS (18985-1-AP), and GAPDH (HRP-60004) were purchased from ProteinTech group (Rosemont, IL); and antibodies against FoxO1 (BS1746) were purchased from bioworld (Bloomington, MN).

Techniques: Control

Journal: Skeletal Muscle

Article Title: Growth differentiation factor 11 induces skeletal muscle atrophy via a STAT3-dependent mechanism in pulmonary arterial hypertension

doi: 10.1186/s13395-022-00292-x

Figure Lengend Snippet: GDF11 levels are accumulated in serum and lung in MCT rats. A GDF11 was measured by ELISA in the serum from rats. B GDF11 expression in lung was detected by Western blot, and GAPDH served as a loading control. C Representative immunohistochemistry of lung sections showing pulmonary arteries stained for GDF11 or CD31 in rats, scale bar is 20 μm. D Western blot analysis of trim63, fbx32, and foxo1 was assessed by Western blot, and protein expression levels were quantified by densitometry. Data are shown as mean ± SEM. Versus vehicle control, * P < 0.05, ** P < 0.01, *** P < 0.001, n = 8 rats/group

Article Snippet: Antibodies against p-STAT3 (Tyr705) (#9145), STAT3 (#9139), p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828), and Smad2/3 (#8685) were purchased from Cell Signaling Technology (Beverly, MA); antibodies against GDF11 (sc-81952) and SOCS3 (sc-51699) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against Fbx32 (ab168372), CD31 (ab28364), and wheat germ agglutinin (ab178444) were purchased from Abcam (Cambridge, MA); recombinant GDF11 protein (1958-GD) were purchased from R&D Systems (Minneapolis, MN); antibodies against Trim63 (55456-1-AP), ubiquitin (10201-2-AP), iNOS (18985-1-AP), and GAPDH (HRP-60004) were purchased from ProteinTech group (Rosemont, IL); and antibodies against FoxO1 (BS1746) were purchased from bioworld (Bloomington, MN).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control, Immunohistochemistry, Staining

Journal: Skeletal Muscle

Article Title: Growth differentiation factor 11 induces skeletal muscle atrophy via a STAT3-dependent mechanism in pulmonary arterial hypertension

doi: 10.1186/s13395-022-00292-x

Figure Lengend Snippet: The GDF11 is involved in myotube atrophy induced by CM from PAEC. A Schematic drawing depicting the generation of CM by hypoxia-culture of PAEC, then myotube was stimulated with CM for 48 h. B Concentrations of GDF11 (pg/mL) in 50% Norm-CM, 20% or 50% Hypo-CM. C Bright-field images of C2C12-derived myotubes treated with either 50% Norm-CM, 20% or 50% Hypo-CM from PAEC, and myotube diameter for conditions represented in the panel. Scale bar is 50 μm. D Myotubes were transfected with GDF11 siRNA or NC siRNA and 50% Norm-CM or 50% Hypo-CM. Protein levels were examined by immunoblotting. E Bright-field images of myotubes treated with 50% Hypo-CM with GDF11 antibody or isotype control, and myotube diameter for conditions represented in the panel. Scale bar is 50μm. F Immunoblots of trim63, fbx32, and foxo1 using lysates from myotubes treated with 50% Norm-CM or 50% Hypo-CM with GDF11 antibody or isotype control; and protein expression levels were quantified by densitometry. Values are presented as average ± SEM. Versus vehicle control, * P < 0.05, ** P < 0.01, *** P < 0.001; versus Hypo-CM control, # P < 0.05, ## P < 0.01, ### P < 0.001; n =3

Article Snippet: Antibodies against p-STAT3 (Tyr705) (#9145), STAT3 (#9139), p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828), and Smad2/3 (#8685) were purchased from Cell Signaling Technology (Beverly, MA); antibodies against GDF11 (sc-81952) and SOCS3 (sc-51699) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against Fbx32 (ab168372), CD31 (ab28364), and wheat germ agglutinin (ab178444) were purchased from Abcam (Cambridge, MA); recombinant GDF11 protein (1958-GD) were purchased from R&D Systems (Minneapolis, MN); antibodies against Trim63 (55456-1-AP), ubiquitin (10201-2-AP), iNOS (18985-1-AP), and GAPDH (HRP-60004) were purchased from ProteinTech group (Rosemont, IL); and antibodies against FoxO1 (BS1746) were purchased from bioworld (Bloomington, MN).

Techniques: Derivative Assay, Transfection, Western Blot, Control, Expressing

Journal: Skeletal Muscle

Article Title: Growth differentiation factor 11 induces skeletal muscle atrophy via a STAT3-dependent mechanism in pulmonary arterial hypertension

doi: 10.1186/s13395-022-00292-x

Figure Lengend Snippet: GDF11 acts via STAT3, SOCS3, and iNOS to induce proteolysis in muscle wasting in vitro. A – D NF-κB, ERK, Smad, or STAT3 dependent luciferase reporters in C2C12 myotubes treated with rGDF11 with the dose ranging from 0 to 100 ng/ml. E Representative Western blots of target proteins (iNOS, phosphorylation and total STAT3, phosphorylation and total Smad2/3, socs3) and loading control (GAPDH) from myotubes treated with rGDF11 with the dose ranging from 0 to 100 ng/ml for 48 h. F NO levels were measured in supernatant from the myotubes described in the panel. G Total protein content of rGDF11-treated myotubes. H Representative western blotting images of ubiquitin from myotubes. I Protein expression of trim63, fbx32, and foxo1 in myotubes treated with rGDF11 with the dose ranging from 0 to 100 ng/ml. GAPDH was used as an internal control. J Bright-field images of myotubes treated with rGDF11, with or without the 26S ribosome inhibitor MG-132 (10 μM) for 48 h; diameter of myotubes for conditions represented in the panel. Scale bar is 50μm. Data presented as mean ± SEM. Versus vehicle control, * P < 0.05, ** P < 0.01, *** P < 0.001; versus rGDF11 control, # P < 0.05, ## P < 0.01, ### P < 0.001; n =3

Article Snippet: Antibodies against p-STAT3 (Tyr705) (#9145), STAT3 (#9139), p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828), and Smad2/3 (#8685) were purchased from Cell Signaling Technology (Beverly, MA); antibodies against GDF11 (sc-81952) and SOCS3 (sc-51699) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against Fbx32 (ab168372), CD31 (ab28364), and wheat germ agglutinin (ab178444) were purchased from Abcam (Cambridge, MA); recombinant GDF11 protein (1958-GD) were purchased from R&D Systems (Minneapolis, MN); antibodies against Trim63 (55456-1-AP), ubiquitin (10201-2-AP), iNOS (18985-1-AP), and GAPDH (HRP-60004) were purchased from ProteinTech group (Rosemont, IL); and antibodies against FoxO1 (BS1746) were purchased from bioworld (Bloomington, MN).

Techniques: In Vitro, Luciferase, Western Blot, Phospho-proteomics, Control, Ubiquitin Proteomics, Expressing

Journal: Skeletal Muscle

Article Title: Growth differentiation factor 11 induces skeletal muscle atrophy via a STAT3-dependent mechanism in pulmonary arterial hypertension

doi: 10.1186/s13395-022-00292-x

Figure Lengend Snippet: Blocking STAT3 activation with Stattic, a STAT3 inhibitor, prevents GDF11 mediated atrophy in vitro. A Bright-field images of myotubes treated with rGDF11 (50ng/ml), with or without STAT3 inhibitor Stattic for 48 h. Scale bars = 50 μm. The fiber widths were measured and calculated (right panel). B Myotubes treated with rGDF11 then with Stattic for 48h were used for Western blot analysis with antibodies against iNOS, pY-STAT3, total STAT3, socs3, and GAPDH. C Total protein content of rGDF11-treated myotubes, with or without STAT3 inhibitor Stattic for 48 h. D NO levels were measured in supernatant from the myotubes described in the panel. E Representative western blotting images of ubiquitin from myotubes. F Protein expression of trim63, fbx32, and foxo1 in myotubes treated with rGDF11 then with Stattic for 48h. G Representative Western blots of phosphorylation and total STAT3 from myotubes treated with rGDF11, siALK5, or AcvRIIb. Data presented as mean ± SEM. Versus vehicle control, * P < 0.05, ** P < 0.01, *** P < 0.001; versus rGDF11 control, # P < 0.05, ## P < 0.01, ### P < 0.001; n =3

Article Snippet: Antibodies against p-STAT3 (Tyr705) (#9145), STAT3 (#9139), p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828), and Smad2/3 (#8685) were purchased from Cell Signaling Technology (Beverly, MA); antibodies against GDF11 (sc-81952) and SOCS3 (sc-51699) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against Fbx32 (ab168372), CD31 (ab28364), and wheat germ agglutinin (ab178444) were purchased from Abcam (Cambridge, MA); recombinant GDF11 protein (1958-GD) were purchased from R&D Systems (Minneapolis, MN); antibodies against Trim63 (55456-1-AP), ubiquitin (10201-2-AP), iNOS (18985-1-AP), and GAPDH (HRP-60004) were purchased from ProteinTech group (Rosemont, IL); and antibodies against FoxO1 (BS1746) were purchased from bioworld (Bloomington, MN).

Techniques: Blocking Assay, Activation Assay, In Vitro, Western Blot, Ubiquitin Proteomics, Expressing, Phospho-proteomics, Control

Journal: Skeletal Muscle

Article Title: Growth differentiation factor 11 induces skeletal muscle atrophy via a STAT3-dependent mechanism in pulmonary arterial hypertension

doi: 10.1186/s13395-022-00292-x

Figure Lengend Snippet: Pathway of STAT3 inhibition in improvement muscle atrophy in the PAH model. A , B The expression of indicated proteins in gastrocnemius muscles was detected by western blot. The band intensities were quantified and total STAT3 or GAPDH was used as control. C Model depicting how STAT3 promotes GDF11-induced muscle wasting. The GDF11 binds to ACVR2B/ALK5 and then activates STAT3 via phosphorylation. Following p-STAT3 translocates to the nucleus and upregulates the expression of iNOS and socs3, leading to the activation of the ubiquitin-proteasome pathway and iNOS/NO pathway, which in turn promotes muscle wasting. Data are shown as mean ± SEM. Versus vehicle control, * P < 0.05, ** P < 0.01, *** P < 0.001; versus MCT control, # P < 0.05, ## P < 0.01, ### P < 0.001; n =3 rats

Article Snippet: Antibodies against p-STAT3 (Tyr705) (#9145), STAT3 (#9139), p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828), and Smad2/3 (#8685) were purchased from Cell Signaling Technology (Beverly, MA); antibodies against GDF11 (sc-81952) and SOCS3 (sc-51699) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against Fbx32 (ab168372), CD31 (ab28364), and wheat germ agglutinin (ab178444) were purchased from Abcam (Cambridge, MA); recombinant GDF11 protein (1958-GD) were purchased from R&D Systems (Minneapolis, MN); antibodies against Trim63 (55456-1-AP), ubiquitin (10201-2-AP), iNOS (18985-1-AP), and GAPDH (HRP-60004) were purchased from ProteinTech group (Rosemont, IL); and antibodies against FoxO1 (BS1746) were purchased from bioworld (Bloomington, MN).

Techniques: Inhibition, Expressing, Muscles, Western Blot, Control, Phospho-proteomics, Activation Assay, Ubiquitin Proteomics