Journal: The Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor β/δ Induces Myogenesis by Modulating Myostatin Activity *

doi: 10.1074/jbc.M111.319145

Figure Lengend Snippet: PPARβ/δ inhibits myostatin activity via Gasp-1. A, top panel, Western blot (IB) analysis of Gasp-1 protein expression in CM collected after 24 h treatment with (+) or without (−) L165041. Middle panels, Western blot analysis of hMstn co-immunoprecipitated (IP) with endogenous hGASP-1 protein present in CM following 24-h treatment with (+) or without (−) L165041. Bottom panel, Ponceau S staining to ensure equal loading of CM. The corresponding graphs show optical density values for hGASP-1 and hMstn protein levels in CM. B, assessment of C2C12 myoblast proliferation, from 0 to 72 h following treatment with CM collected from L165041 and control-treated (DMSO) myoblasts as monitored by methylene blue staining. C, assessment of human myoblast proliferation at 72 h following treatment with DMSO (Control) or L165041 in the absence (PBS) or presence (+Ab) of 1 μg/ml anti-hGASP-1 antibody as monitored by methylene blue assay. D, assessment of C2C12 myoblast proliferation at 48 h following treatment with CM collected from PPARβ/δ-null and wild type primary myoblast cultures as monitored by methylene blue assay. E, assessment of PPARβ/δ-null and wild type mice primary myoblast proliferation at 24 and 48 h following treatment with dialysis buffer (DB) or sActRIIB (2 μg/ml) protein as monitored by methylene blue assay. F, assessment of SBE-4x-Luc reporter activity in C2C12 myoblasts treated with CM collected from L165041 or control-treated (DMSO) C2C12 myoblasts; in primary myoblast cultures derived from PPARβ/δ-null and wild type mice; and in C2C12 cells treated with dialysis buffer or with sActRIIB protein (2 μg/ml). All SBE-4x-Luc reporter-transfected cultures were grown for 24 h under proliferating conditions prior to collection. The corresponding graph represents the -fold change in luciferase activity normalized to Renilla luciferase. Each bar represents the mean ± S.E. of triplicate samples from two independent experiments. G, qPCR analysis of Mstn mRNA expression in L165041 and control-treated (DMSO) C2C12 myoblasts. The graph represents -fold change normalized to GAPDH expression. Data are mean ± S.E. (n = 3). H, Western blot analysis of Mstn protein expression in gastrocnemius muscle isolated from PPARβ/δ-null and wild type mice. The corresponding graph (right) shows the optical density values of Mstn protein expression in PPARβ/δ-null and wild type mice. α-Tubulin expression was analyzed to ensure equal loading of samples. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The antibodies used in this study are as follows: rabbit polyclonal anti-MyoD (C-20) (sc-304, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-myogenin (M-225) (sc-576, Santa Cruz Biotechnology); mouse monoclonal anti-MyHC, all types (MF-20 C, Developmental Studies Hybridoma Bank, Iowa City, IA); mouse monoclonal anti-PPARβ/δ (F-10) X (sc-74517, Santa Cruz Biotechnology); mouse monoclonal anti-hGASP-1 (MAB2070, R&D Systems); rabbit polyclonal anti-human myostatin antibody (HPA021681, Sigma-Aldrich); rat monoclonal anti-mouse myostatin antibody (sc-74041, Santa Cruz Biotechnology); and purified mouse monoclonal anti α-tubulin antibody (T-9026, Sigma-Aldrich).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Staining, Control, Derivative Assay, Transfection, Luciferase, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor β/δ Induces Myogenesis by Modulating Myostatin Activity *

doi: 10.1074/jbc.M111.319145

Figure Lengend Snippet: PPARβ/δ activation modulates myostatin activity via Gasp-1 during postnatal myogenesis. Both exogenous (L165041) and endogenous skeletal muscle ligands (such as released during exercise and muscle wasting) signal to activate PPARβ/δ. Activated PPARβ/δ heterodimerizes with the co-activator RXR and binds to the PPRE (DR-1) in the Gasp-1 gene to facilitate up-regulation of Gasp-1 expression. Gasp-1 undergoes posttranscriptional modification to add secretory signals prior to being secreted into circulation. The secreted Gasp-1 can then interact with Mstn and block further signaling of myostatin through its receptors, which we propose results in the enhanced skeletal muscle myogenesis observed following activation of PPARβ/δ.

Article Snippet: The antibodies used in this study are as follows: rabbit polyclonal anti-MyoD (C-20) (sc-304, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-myogenin (M-225) (sc-576, Santa Cruz Biotechnology); mouse monoclonal anti-MyHC, all types (MF-20 C, Developmental Studies Hybridoma Bank, Iowa City, IA); mouse monoclonal anti-PPARβ/δ (F-10) X (sc-74517, Santa Cruz Biotechnology); mouse monoclonal anti-hGASP-1 (MAB2070, R&D Systems); rabbit polyclonal anti-human myostatin antibody (HPA021681, Sigma-Aldrich); rat monoclonal anti-mouse myostatin antibody (sc-74041, Santa Cruz Biotechnology); and purified mouse monoclonal anti α-tubulin antibody (T-9026, Sigma-Aldrich).

Techniques: Activation Assay, Activity Assay, Expressing, Modification, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor β/δ Induces Myogenesis by Modulating Myostatin Activity *

doi: 10.1074/jbc.M111.319145

Figure Lengend Snippet: PPARβ/δ inhibits myostatin activity via Gasp-1. A, top panel, Western blot (IB) analysis of Gasp-1 protein expression in CM collected after 24 h treatment with (+) or without (−) L165041. Middle panels, Western blot analysis of hMstn co-immunoprecipitated (IP) with endogenous hGASP-1 protein present in CM following 24-h treatment with (+) or without (−) L165041. Bottom panel, Ponceau S staining to ensure equal loading of CM. The corresponding graphs show optical density values for hGASP-1 and hMstn protein levels in CM. B, assessment of C2C12 myoblast proliferation, from 0 to 72 h following treatment with CM collected from L165041 and control-treated (DMSO) myoblasts as monitored by methylene blue staining. C, assessment of human myoblast proliferation at 72 h following treatment with DMSO (Control) or L165041 in the absence (PBS) or presence (+Ab) of 1 μg/ml anti-hGASP-1 antibody as monitored by methylene blue assay. D, assessment of C2C12 myoblast proliferation at 48 h following treatment with CM collected from PPARβ/δ-null and wild type primary myoblast cultures as monitored by methylene blue assay. E, assessment of PPARβ/δ-null and wild type mice primary myoblast proliferation at 24 and 48 h following treatment with dialysis buffer (DB) or sActRIIB (2 μg/ml) protein as monitored by methylene blue assay. F, assessment of SBE-4x-Luc reporter activity in C2C12 myoblasts treated with CM collected from L165041 or control-treated (DMSO) C2C12 myoblasts; in primary myoblast cultures derived from PPARβ/δ-null and wild type mice; and in C2C12 cells treated with dialysis buffer or with sActRIIB protein (2 μg/ml). All SBE-4x-Luc reporter-transfected cultures were grown for 24 h under proliferating conditions prior to collection. The corresponding graph represents the -fold change in luciferase activity normalized to Renilla luciferase. Each bar represents the mean ± S.E. of triplicate samples from two independent experiments. G, qPCR analysis of Mstn mRNA expression in L165041 and control-treated (DMSO) C2C12 myoblasts. The graph represents -fold change normalized to GAPDH expression. Data are mean ± S.E. (n = 3). H, Western blot analysis of Mstn protein expression in gastrocnemius muscle isolated from PPARβ/δ-null and wild type mice. The corresponding graph (right) shows the optical density values of Mstn protein expression in PPARβ/δ-null and wild type mice. α-Tubulin expression was analyzed to ensure equal loading of samples. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The antibodies used in this study are as follows: rabbit polyclonal anti-MyoD (C-20) (sc-304, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-myogenin (M-225) (sc-576, Santa Cruz Biotechnology); mouse monoclonal anti-MyHC, all types (MF-20 C, Developmental Studies Hybridoma Bank, Iowa City, IA); mouse monoclonal anti-PPARβ/δ (F-10) X (sc-74517, Santa Cruz Biotechnology); mouse monoclonal anti-hGASP-1 (MAB2070, R&D Systems); rabbit polyclonal anti-human myostatin antibody (HPA021681, Sigma-Aldrich); rat monoclonal anti-mouse myostatin antibody (sc-74041, Santa Cruz Biotechnology); and purified mouse monoclonal anti α-tubulin antibody (T-9026, Sigma-Aldrich).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Staining, Derivative Assay, Transfection, Luciferase, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor β/δ Induces Myogenesis by Modulating Myostatin Activity *

doi: 10.1074/jbc.M111.319145

Figure Lengend Snippet: PPARβ/δ activation modulates myostatin activity via Gasp-1 during postnatal myogenesis. Both exogenous (L165041) and endogenous skeletal muscle ligands (such as released during exercise and muscle wasting) signal to activate PPARβ/δ. Activated PPARβ/δ heterodimerizes with the co-activator RXR and binds to the PPRE (DR-1) in the Gasp-1 gene to facilitate up-regulation of Gasp-1 expression. Gasp-1 undergoes posttranscriptional modification to add secretory signals prior to being secreted into circulation. The secreted Gasp-1 can then interact with Mstn and block further signaling of myostatin through its receptors, which we propose results in the enhanced skeletal muscle myogenesis observed following activation of PPARβ/δ.

Article Snippet: The antibodies used in this study are as follows: rabbit polyclonal anti-MyoD (C-20) (sc-304, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-myogenin (M-225) (sc-576, Santa Cruz Biotechnology); mouse monoclonal anti-MyHC, all types (MF-20 C, Developmental Studies Hybridoma Bank, Iowa City, IA); mouse monoclonal anti-PPARβ/δ (F-10) X (sc-74517, Santa Cruz Biotechnology); mouse monoclonal anti-hGASP-1 (MAB2070, R&D Systems); rabbit polyclonal anti-human myostatin antibody (HPA021681, Sigma-Aldrich); rat monoclonal anti-mouse myostatin antibody (sc-74041, Santa Cruz Biotechnology); and purified mouse monoclonal anti α-tubulin antibody (T-9026, Sigma-Aldrich).

Techniques: Activation Assay, Activity Assay, Expressing, Modification, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Evaluation of the In Vitro Damage Caused by Lipid Factors on Stem Cells from a Female Rat Model of Type 2 Diabetes/Obesity and Stress Urinary Incontinence

doi: 10.3390/ijms21145045

Figure Lengend Snippet: ZF4-SC were only mildly induced by cholesterol, or not by Na palmitate, to overexpress myostatin, a lipofibrotic effector, and not affected for the expression of PCNA, a proliferation mediator. ZF4-SC were incubated in triplicate, as in , with or without cholesterol or Na palmitate addition, but until day 4 close to confluence. Then cells were collected for Western blot analysis, probing sequentially with antibodies against myostatin, β -actin as a housekeeping protein, and PCNA, followed by the respective secondary antibodies and luminol. The bands for myostatin (MSTN) (50, 32, and 25 kDa) are shown on top. The ones for β -actin (42 kDa) and PCNA (36 kDa) from the re-probing and re-exposures are below them. At the bottom: densitometry graphs for MSTN and PCNA, corrected by β- actin.; * p < 0.05 (CHOL vs. CTRL).

Article Snippet: Immuno-detection on the membranes was with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) against myostatin (GDF8/11, mouse monoclonal antibody SC-393335); and housekeeping beta-actin (mouse monoclonal, followed by secondary antibodies: anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology, Danvers, MA, USA), or anti-rabbit IgG linked to HRP (Amersham GE, Pittsburgh, PA, USA).

Techniques: Expressing, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Evaluation of the In Vitro Damage Caused by Lipid Factors on Stem Cells from a Female Rat Model of Type 2 Diabetes/Obesity and Stress Urinary Incontinence

doi: 10.3390/ijms21145045

Figure Lengend Snippet: Despite the observed mild overexpression of myostatin protein by cholesterol and its lack of induction by Na palmitate in ZF4-SC, the miRs related to myostatin, were also inhibited by the lipid factors. The current results are shown on the right two columns, for RNA isolated from the standard 4 days incubations of ZF4-SC with Na palmitate and cholesterol or no addition, which were assayed for their miR-global transcriptional signatures (miR-GTS). Only individual miRs related to myostatin expression were selected for tabulation, previously arranged by their level of expression on male MDSC exposed in vivo to type 2 diabetes/obesity (T2D/O) [ 7 ] on LD-MDSC vs. non T2D/O in ED- MDSC. Blue highlighting for current results shows downregulation < 0.50 against controls. Yellow highlighting is similarly applied to the same miRs previously reported both in male rats in vivo [ 7 ] and in vitro with dyslipidemic Obese Zucker (OZ) serum in female rats [ 8 ], and now are included as a reference. Green highlighting is for the miRs, which previously were shown to be in agreement with the in vitro results with dyslipidemic serum in female ZF4-SC.

Article Snippet: Immuno-detection on the membranes was with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) against myostatin (GDF8/11, mouse monoclonal antibody SC-393335); and housekeeping beta-actin (mouse monoclonal, followed by secondary antibodies: anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology, Danvers, MA, USA), or anti-rabbit IgG linked to HRP (Amersham GE, Pittsburgh, PA, USA).

Techniques: Over Expression, Isolation, Expressing, In Vivo, In Vitro, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Evaluation of the In Vitro Damage Caused by Lipid Factors on Stem Cells from a Female Rat Model of Type 2 Diabetes/Obesity and Stress Urinary Incontinence

doi: 10.3390/ijms21145045

Figure Lengend Snippet: A similar decrease of key miRs unrelated to myostatin by lipid factors was found in ZF4-SC. See Table 1 for the legend explanation, except that here the selected miRs were unrelated to myostatin. Within those selected miRs were the ones most expressed and changed by incubation as reported previously [ 8 ]. Purple highlighting shows the only two in vivo, which within the selected miRs were upregulated.

Article Snippet: Immuno-detection on the membranes was with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) against myostatin (GDF8/11, mouse monoclonal antibody SC-393335); and housekeeping beta-actin (mouse monoclonal, followed by secondary antibodies: anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology, Danvers, MA, USA), or anti-rabbit IgG linked to HRP (Amersham GE, Pittsburgh, PA, USA).

Techniques: Incubation, In Vivo, In Vitro, Comparison

Journal: Journal of stem cell research & therapy

Article Title: Muscle Derived Stem Cells Stimulate Muscle Myofiber Repair and Counteract Fat Infiltration in a Diabetic Mouse Model of Critical Limb Ischemia

doi: 10.4172/2157-7633.1000370

Figure Lengend Snippet: The treatment of the diabetic CLI muscle with MDSC or with concurrent molsidomine upregulated the muscle mass inhibitor and profibrotic factor myostatin, but did not change the levels of its antagonist, follistatin. A: fresh ischemic or normal gastrocnemius tissue was subjected to quantitative western blot analysis of myostatin, corrected by GAPDH; two representative specimens per group are shown (total n=8/group). B: as in the top panel, but for follistatin. The higher the myostatin/follistatin ratio the higher the profibrotic and muscle mass inhibition effects. ND-UT: non-diabetic, non-UFAL, untreated; UT: UFAL, untreated; SC: UFAL treated with MDSC alone; SC+Mol: UFAL treated with MDSC and molsidomine; Mol: UFAL treated with molsidomine alone. *p<0.05; **p<0.01; ***p<0.001, Asterisks refer exclusively to comparisons performed against the untreated group (UT) subjected to UFAL as control. The absence of asterisks indicates non-significance against the UT group subjected to UFAL.

Article Snippet: Blots were probed with primary antibodies: (a) Oct 4 (anti-rabbit Oct 4, polyclonal, 1:500; BioVision, Inc., Milpitas, California, U.S.A.) [ ]; (b) ASMA (anti-human ASMA, monoclonal, 1:1,000; Calbiochem, Billerica, Massachusetts, USA) [ ]; (c) Calponin I (anti-mouse calponin 1, monoclonal, 1:500; Santa Cruz Biotechnology, Dallas, Texas, U.S.A) [ ]; (d) VEGF (anti-mouse VEGF, monoclonal, 1:500; Santa Cruz Biotechnology) [ , ]; (e) Von Willebrand (anti-mouse Von Willebrand, polyclonal, 1:500; Abcam, Cambridge, Massachusetts, U.S.A.) [ ]; (f) CD31 (anti-mouse CD31, monoclonal, 1:1000, Abcam) [ ]; (g) BDNF (anti-mouse BDNF, rabbit monoclonal, 1:1000; Abcam) [ ]; (h) MHC (fast) (anti-mouse MHC-II, monoclonal, 1:5000; Abcam) [ ]; (i) myostatin (anti-mouse myostatin, rabbit polyclonal 1:1,000; Chemicon International Inc, Billerica, Massachusetts, U.S.A.) [ , ]; (j) follistatin (anti-mouse follistatin, rabbit polyclonal, 1:1000, Abcam) [ ]; and (k) GAPDH (anti-mouse GAPDH, monoclonal, 1:5,000, Chemicon International).

Techniques: Western Blot, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor β/δ Induces Myogenesis by Modulating Myostatin Activity

doi: 10.1074/jbc.m111.319145

Figure Lengend Snippet: FIGURE 5. PPAR/ inhibits myostatin activity via Gasp-1. A, top panel, Western blot (IB) analysis of Gasp-1 protein expression in CM collected after 24 h treatment with () or without () L165041. Middle panels, Western blot analysis of hMstn co-immunoprecipitated (IP) with endogenous hGASP-1 protein present in CM following 24-h treatment with () or without () L165041. Bottom panel, Ponceau S staining to ensure equal loading of CM. The corresponding graphs show optical density values for hGASP-1 and hMstn protein levels in CM. B, assessment of C2C12 myoblast proliferation, from 0 to 72 h following treatmentwithCMcollectedfromL165041andcontrol-treated(DMSO)myoblastsasmonitoredbymethylenebluestaining.C,assessmentofhumanmyoblast proliferation at 72 h following treatment with DMSO (Control) or L165041 in the absence (PBS) or presence (Ab) of 1 g/ml anti-hGASP-1 antibody as monitored by methylene blue assay. D, assessment of C2C12 myoblast proliferation at 48 h following treatment with CM collected from PPAR/-null and wild type primary myoblast cultures as monitored by methylene blue assay. E, assessment of PPAR/-null and wild type mice primary myoblast proliferation at 24 and 48 h following treatment with dialysis buffer (DB) or sActRIIB (2 g/ml) protein as monitored by methylene blue assay. F, assessment of SBE-4x-Luc reporter activity in C2C12 myoblasts treated with CM collected from L165041 or control-treated (DMSO) C2C12 myoblasts; in primary myoblast cultures derived from PPAR/-null and wild type mice; and in C2C12 cells treated with dialysis buffer or with sActRIIB protein (2 g/ml). All SBE-4x-Luc reporter-transfected cultures were grown for 24 h under proliferating conditions prior to collection. The corresponding graph represents the -fold change in luciferase activity normalized to Renilla luciferase. Each bar represents the mean S.E. of triplicate samples from two independent experiments. G, qPCR analysis of Mstn mRNA expression in L165041 and control-treated (DMSO) C2C12 myoblasts. The graph represents -fold change normalized to GAPDH expression. Data are mean S.E. (n 3). H, Western blot analysis of Mstn protein expression in gastrocnemius muscle isolated from PPAR/-null and wild type mice. The corresponding graph (right) shows the optical density values of Mstn protein expression in PPAR/-null and wild type mice. -Tubulin expression was analyzed to ensure equal loading of samples. *, p 0.05; **, p 0.01; ***, p 0.001.

Article Snippet: The antibodies used in this study are as follows: rabbit polyclonal anti-MyoD (C-20) (sc-304, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-myogenin (M-225) (sc-576, Santa Cruz Biotechnology); mouse monoclonal anti-MyHC, all types (MF-20 C, Developmental Studies Hybridoma Bank, Iowa City, IA); mouse monoclonal anti-PPAR / (F-10) X (sc-74517, Santa Cruz Biotechnology); mouse monoclonal anti-hGASP-1 (MAB2070, R&D Systems); rabbit polyclonal anti-human myostatin antibody (HPA021681, Sigma-Aldrich); rat monoclonal anti-mouse myostatin antibody (sc-74041, Santa Cruz Biotechnology); and purified mouse monoclonal anti -tubulin antibody (T-9026, Sigma-Aldrich).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Staining, Control, Derivative Assay, Transfection, Luciferase, Isolation

Journal: Journal of Biological Chemistry

Article Title: Peroxisome Proliferator-activated Receptor β/δ Induces Myogenesis by Modulating Myostatin Activity

doi: 10.1074/jbc.m111.319145

Figure Lengend Snippet: FIGURE 8. PPAR/ activation modulates myostatin activity via Gasp-1 during postnatal myogenesis. Both exogenous (L165041) and endogenous skeletal muscle ligands (such as released during exercise and muscle wasting) signal to activate PPAR/. Activated PPAR/ heterodimerizes with the co-activator RXR and binds to the PPRE (DR-1) in the Gasp-1 gene to facilitate up-regulation of Gasp-1 expression. Gasp-1 undergoes posttranscriptional modification to add secretory signals prior to being secreted into circulation. The secreted Gasp-1 can then interact with Mstn and block further signaling of myostatin through its receptors, which we propose results in the enhanced skeletal muscle myogenesis observed following activation of PPAR/.

Article Snippet: The antibodies used in this study are as follows: rabbit polyclonal anti-MyoD (C-20) (sc-304, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-myogenin (M-225) (sc-576, Santa Cruz Biotechnology); mouse monoclonal anti-MyHC, all types (MF-20 C, Developmental Studies Hybridoma Bank, Iowa City, IA); mouse monoclonal anti-PPAR / (F-10) X (sc-74517, Santa Cruz Biotechnology); mouse monoclonal anti-hGASP-1 (MAB2070, R&D Systems); rabbit polyclonal anti-human myostatin antibody (HPA021681, Sigma-Aldrich); rat monoclonal anti-mouse myostatin antibody (sc-74041, Santa Cruz Biotechnology); and purified mouse monoclonal anti -tubulin antibody (T-9026, Sigma-Aldrich).

Techniques: Activation Assay, Activity Assay, Expressing, Modification, Blocking Assay