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 regulates myoblast proliferation and differentiation. A, proliferation analysis of C2C12 myoblast cultures grown under proliferating conditions in the absence (Control (DMSO)) or presence of the PPARβ/δ agonist L165041 (10 μm) for 96 h as monitored by a methylene blue assay. B, representative images of H&E-stained differentiating myoblasts across a differentiation time course (24–96 h) in L165041 and control-treated myoblasts. Scale bars, 100 μm. C, quantification of myotube number from 12 random images/coverslip (n = 3) from three independent experiments. D, quantification of fusion index in C2C12 myotubes cultures at 48 and 72 h of differentiation in the absence (Control) or presence of L165041. The graph shows the mean percentage fusion index ± S.E. over three coverslips/treatment. E, frequency distribution of myotube area (μm2) at 96 h of differentiation in L165041 and control-treated myotubes as calculated from 12 random images/coverslip (n = 3). F and G, qPCR analysis of MyoD (F) and myogenin (G) mRNA expression during differentiation (0–96 h) in L165041 and control-treated C2C12 myoblasts. The graphs represent -fold change normalized to GAPDH. Data are mean ± S.E. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Mouse C2C12 myoblasts ( 32 ) were obtained from American Type Culture Collection (Manassas, VA) and maintained as described previously ( 33 ).

Techniques: Control, Staining, Expressing

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: Microarray analysis, data validation, and identification of Gasp-1 as a novel PPARβ/δ downstream target gene. A, gene expression changes in 72-h differentiated C2C12 myotubes in response to treatment with L165041 (10 μm) for a further 0, 1, 4, 6, 8, 12, and 24 h. Left panel, shows a heat map representing genes that are differentially expressed upon L165041 treatment across the time course (p ≤ 0.05; two-way ANOVA). Each condition is representative of data from three samples; the color scheme, which represents the -fold change, is indicated at the bottom of heat map. Right panel, qPCR analysis of the mRNA expression of 24 selected genes from the microarray analysis. The data shows -fold change ± S.E. normalized to GAPDH from three independent experiments. The normalized gene expression data were converted into a heat map using Orange 2.0b-data mining, fruitful and fun software. B and C, qPCR analysis of Gasp-1 mRNA expression in C2C12 myoblasts during proliferation (0, 8, 12, and 24 h) (B) and across a differentiation time course (0–96 h) (C). Corresponding graphs show fold change ± S.E. normalized to GAPDH from three independent experiments. D, Western blot analysis of hGASP-1 protein levels in CM from L165041 and control-treated (DMSO) human myoblasts. hGASP-1 protein was immunoprecipitated using a mouse monoclonal anti-hGASP-1 antibody prior to Western blot analysis. The corresponding graph shows optical density values of hGASP-1 protein in CM. E, Western blot analysis of hGASP-1 protein expression in human myoblasts across a differentiation time course (24–96 h) in the presence (+) or absence (−) of L165041. α-Tubulin expression was analyzed to ensure equal loading of samples. The graph represents optical density values for hGASP-1 protein levels. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Mouse C2C12 myoblasts ( 32 ) were obtained from American Type Culture Collection (Manassas, VA) and maintained as described previously ( 33 ).

Techniques: Microarray, Biomarker Discovery, Gene Expression, Expressing, Software, Western Blot, Control, Immunoprecipitation

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: Ligand-mediated activation of PPARβ/δ induces Gasp-1 promoter-reporter luciferase activity. A, qPCR analysis of Gasp-1 mRNA expression in EDL, gastrocnemius (GAS), soleus, and quadriceps (Quad) muscles from PPARβ/δ-null and wild type mice. Data are mean ± S.E. (n = 3). The graphs represent -fold change normalized to GAPDH expression. B, qPCR analysis of Gasp-1 mRNA expression in differentiating primary myoblasts isolated from 10-week-old PPARβ/δ-null and wild type mice. The graph represents -fold change normalized to GAPDH expression. Data are mean ± S.E. (n = 3). C and D, qPCR analysis of PPARβ/δ (C) and Gasp-1 (D) mRNA expression in EDL muscle isolated from 10-week-old mdx and wild type mice. Data are mean ± S.E. (n = 3). The graphs represent -fold change normalized to GAPDH expression. E, localization of the consensus DR-1 site present within the 1.5-kb proximal Gasp-1 promoter region. F, homology between the mouse and human DR-1 motif present within the 1.5-kb proximal Gasp-1 promoter region. G, top, schematic representation of reporter constructs used for luciferase analysis. Bottom, assessment of promoter-luciferase reporter activity in C2C12 myoblasts transfected with the empty vector control (pGL3-basic), the 1.5-kb proximal Gasp-1 luciferase construct (pGL3-Gasp-1), or the 1-kb DR-1 deletion construct (pGL3-Gasp-1 del) following 24-h treatment with DMSO (Control), L165041 (10 μm), GW1929 (30 μm), or Wy14643 (10 μm). H, top, sequences highlighting the DR-1 site (bold) in both wild type (wt-Gasp-1) and mutated Gasp-1 promoters (mut-Gasp-1). Arrows indicate the mutated base pairs. Bottom, assessment of promoter-luciferase reporter activity in C2C12 myoblasts transfected with empty vector control (pGL3-basic), pGL3-Gasp-1, or mut-pGL3-Gasp-1, following treatment for 24 h with DMSO (Control), L165041 (10 μm), GW1929 (30 μm), or Wy14643 (10 μm). Promoter-reporter luciferase activity was normalized to Renilla luciferase and expressed as -fold change relative to the empty vector control (pGL3-basic). Each bar represents the mean values ± S.E. from four independent experiments. I, agarose gel image revealing the interaction of PPARβ/δ with the DR-1 site of the Gasp-1 promoter in the absence (−) or presence (+) of L165041 as assessed through ChIP (upper panel). Analysis of PPARβ/δ interaction with the β-actin promoter in the absence (−) or presence (+) of L165041 was also performed as a negative control (lower panel). The relative amounts of both the Gasp-1 and β-actin promoters in the input were also assessed and are indicated. Both isotype-specific IgG and no antibody (No Ab) controls are shown. *, p < 0.05; **, p < 0.01.

Article Snippet: Mouse C2C12 myoblasts ( 32 ) were obtained from American Type Culture Collection (Manassas, VA) and maintained as described previously ( 33 ).

Techniques: Activation Assay, Luciferase, Activity Assay, Expressing, Muscles, Isolation, Construct, Transfection, Plasmid Preparation, Control, Agarose Gel Electrophoresis, Negative Control

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: Mouse C2C12 myoblasts ( 32 ) were obtained from American Type Culture Collection (Manassas, VA) and maintained as described previously ( 33 ).

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: hGASP-1 enhances human myoblast proliferation and differentiation. A, proliferation analysis of human myoblasts cultured for 48 h in the absence (0.01% BSA) or presence of various concentrations (0.5, 1, 2, and 5 μg/ml) of rhGASP-1 as measured by methylene blue assay. B, representative images of H&E-stained 72-h differentiated human myotube cultures in the absence (0.01% BSA) or presence of rhGASP-1 protein. Scale bars, 100 μm. C, quantification of myotube number in 72-h differentiated human myotubes cultures in the absence (0.01% BSA) or presence of rhGASP-1 protein from 12 random images/coverslip (n = 3). D, frequency distribution of myotube area (μm2) at 72 h of differentiation in rhGASP-1 protein-treated and control-treated (0.01% BSA) human myotube cultures as calculated from 12 random images/coverslip (n = 3). E, assessment of SBE-4x-Luc reporter activity in C2C12 myoblasts grown for 24 h in the absence (−) or presence of either 1:4 (+) or 1:2 (++) diluted CM from Mstn protein-secreting CHO cells. SBE-4x-Luc reporter activity was also assessed following treatment without (−) or with increasing concentrations (0.5, 1, and 2 μg/ml) of rhGASP-1. 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. *, p < 0.05; **, p < 0.01.

Article Snippet: Mouse C2C12 myoblasts ( 32 ) were obtained from American Type Culture Collection (Manassas, VA) and maintained as described previously ( 33 ).

Techniques: Cell Culture, Staining, Control, Activity Assay, Luciferase

Journal: Immunity

Article Title: Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual

doi: 10.1016/j.immuni.2019.02.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: L-a-Phosphatidylinositol-4-phosphate extracted from porcine brain (PIP) , Avanti Polar Lipids , Cat# 840045P.

Techniques: Purification, Virus, Clinical Proteomics, Recombinant, Staining, Plasmid Preparation, Luciferase, Cell Culture, Lysis, Reporter Gene Assay, Protease Inhibitor, Modification, Library Amplification, Binding Assay, Transfection, Crystallization Assay, Sequencing, Expressing, Software, Biomarker Discovery