Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: Recombinant MSTN suppressed AChR clustering and miR‐206 expression. (a) AChR and MyHC staining of MuSCs cultures treated with or without recombinant MSTN (100 ng/mL) and follistatin (500 ng/mL). Scale bar = 50μm. (b) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. (c) The levels of miR‐206 expression in MuSCs cultures. Mature myotubes differentiated from MAS‐derived MuSCs were treated with control, agrin alone, agrin plus MSTN or agrin plus MSTN and follistatin. (d) AChR and MyHC staining of myotubes transfected with miR‐206 mimics or negative control (NC) prior to treatment with agrin and MSTN. Scale bar = 50 μm. (e) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mature myotubes were treated with 100 ng/mL agrin (HY‐ P79236 , MedChemExpress, China) in DM or conditioned medium (CM) of 7 dpi FAPs supplemented with 2% HS for 16 h. For recombinant MSTN treatment, 100 ng/mL MSTN (HY‐ P72632 , MedChemExpress, China) with or without 500 ng/mL follistatin (HY‐ P70315 , MedChemExpress, China) was administered together with agrin treatment.

Techniques: Recombinant, Expressing, Staining, Derivative Assay, Control, Transfection, Negative Control

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Pid1 induces insulin resistance in both human and mouse skeletal muscle during obesity.

doi: 10.1210/me.2013-1048

Figure Lengend Snippet: Figure 5. Myostatin Signals via NF-B to Up-Regulate Pid1 Gene Expression. A, Schematic representation of human PID1 (1.3 kb) and mouse Pid1 (4.5-kb) upstream elements. Locations of putative Sp1, GATA, STRE, and NF-B binding elements, as identified through in silico analysis, are indicated. Arrows denote transcription start sites in both human and mouse PID1/Pid1 promoters. B (upper panel), Schematic representation of the 0.978-kb proximal human PID1 promoter-reporter construct (PID1 promoter) used for subsequent analysis. The locations of the putative NF-B binding elements are indicated. B (lower panel), Assessment of PID1 promoter-reporter luciferase activity in C2C12 myoblasts cotransfected with either empty vector control (pLightSwitch-Prom) or PID1 promoter (pLightSwitch-PID1), and treated with CHO-control () or a 1:4 ( ) or 1:2 ( ) dilution of CHO-Mstn. Luciferase activity was normalized to firefly luciferase activity and expressed as relative luciferase activity. Bars represent relative luciferase activity SEM and are representative of triplicate experiments. P .01 (**). C, Assessment of PID1 promoter-reporter luciferase activity in C2C12 myoblasts transfected with either pLightSwitch-Prom or pLightSwitch-PID1 promoter and further treated with the absence () or presence ( ) of Mstn-specific siRNA (Mstn siRNA). Luciferase activity was normalized to firefly luciferase activity and expressed as relative luciferase activity. Bars represent relative luciferase activity SEM and are representative of triplicate experiments. P .01 (**). D, Assessment of PID1 promoter- reporter luciferase activity in C2C12 myoblasts transfected with either pLightSwitch-Pro or pLightSwitch-PID1 and treated with ( ) or without () a 1:4 dilution of CHO-Mstn in the absence () or presence ( ) of the NF-B-specific inhibitor BAY-117085. Luciferase activity was normalized to firefly luciferase activity and expressed as relative luciferase activity. Bars represent relative luciferase activity SEM and are representative of triplicate experiments. P .01 (**) and P .001 (***). E, ChIP analysis of NF-B interaction with NF-B binding elements in the human PID1 promoter and further treated with CHO-control () or CHO- Mstn ( ) in the absence () or presence ( ) of BAY-117085. PCR amplicons for input Genomic DNA Input-NF-B (PID1), negative control antibody (anti-IgG), and anti-NF-B (p65) are indicated.

Article Snippet: The pGL3P (catalog E1761) firefly luciferase control vector was purchased from Promega Corp. Mstn-specific shRNA plasmids (catalog no. TG512805) were obtained from Origene.

Techniques: Gene Expression, Binding Assay, In Silico, Construct, Luciferase, Activity Assay, Plasmid Preparation, Control, Transfection, Negative Control

Journal: Frontiers in Genetics

Article Title: Beyond the Big Five: Investigating Myostatin Structure, Polymorphism and Expression in Camelus dromedarius

doi: 10.3389/fgene.2019.00502

Figure Lengend Snippet: Amino acidic sequence of the C. dromedarius myostatin as inferred from the cDNA sequence. (A) Schematic outline. The three protein domains (signal peptide, pro-region, and active peptide) are highlighted in different colors (yellow, blue, and green, respectively). The two most likely residues involved in the signal peptide cleavage (see main text) are indicated by black arrows. Similarly, the residue (D, for aspartic acid) shown to be essential for BMP/tolloid protease cleavage, and the motif (RSRR) needed for recognition by furin convertase, are highlighted. (B) Amino acidic sequence of the C. dromedarius myostatin, with the three protein domains highlighted in different colors, as in (A) . The above mentioned hallmarks are also depicted here (signal peptide cleavage, black arrows; BMP/tolloid protease cleavage residue and furin convertase recognition motif, bold). In addition, the nine conserved cysteine residues in the active peptide are indicated (bold and white).

Article Snippet: The used primary antibody was a rabbit polyclonal anti-MSTN antibody against the C-terminal region (300–349 aa) of mouse myostatin (TA343358, OriGene; dilution 1:1000) that presented broad species reactivity, including artiodactyls.

Techniques: Sequencing, Residue

Journal: Frontiers in Genetics

Article Title: Beyond the Big Five: Investigating Myostatin Structure, Polymorphism and Expression in Camelus dromedarius

doi: 10.3389/fgene.2019.00502

Figure Lengend Snippet: Comparative analysis of myostatin protein sequences. (A) The alignment of myostatin orthologous sequences sampled from various mammalian species is presented. Blue arrowheads indicate the variants detected between the four considered Camelidae sequences and the five Bovidae sequences at 13 specific sites. Black arrowheads indicate the variants detected between the four considered Camelidae sequences and the Homo sapiens sequence at 5 specific sites, different from the sites previously cited. The gray arrowhead indicates the position of two different variants detected in H. sapiens and in Bovidae in correspondence of Q164 from C. dromedarius . Orange “boxes” indicate variations between the H. sapiens sequence retrieved from refseq_database and the sequence of the human crystallized myostatin. Amino acid codes and numbering refers to the C. dromedarius myostatin. (B) Lateral view of the 3D comparative model of the C. dromedarius myostatin dimer. The protein is reported in green/magenta cartoon representation. Variants observed between Camelidae myostatin sequences and human/Bovidae myostatin are reported in black (5)/blue (13) spheres in chain A, and dark-yellow (5)/cyan (13) spheres in chain B, respectively. The only site of C. dromedarius myostatin showing a variation both in H. sapiens and in Bovidae locates at site 164 (Q164 for C. dromedarius , E164 in H. sapiens , K164 in B. taurus ) and is indicated by gray spheres. Notably, variants observed between C. dromedarius and Bovidae occur at different sites with respect to those detected between C. dromedarius and H. sapiens , with the exclusion of residues at site 164. Residues C339/C340 of chain A and chain B, forming inter-monomer disulphide bridges, are reported in red spheres. R65 of chain A and chain B, involved in interactions with T114, is indicated by red sticks.

Article Snippet: The used primary antibody was a rabbit polyclonal anti-MSTN antibody against the C-terminal region (300–349 aa) of mouse myostatin (TA343358, OriGene; dilution 1:1000) that presented broad species reactivity, including artiodactyls.

Techniques: Sequencing

Journal: Frontiers in Genetics

Article Title: Beyond the Big Five: Investigating Myostatin Structure, Polymorphism and Expression in Camelus dromedarius

doi: 10.3389/fgene.2019.00502

Figure Lengend Snippet: Phylogenetic analysis of myostatin protein sequences. Numbers indicate bootstrap values higher than 60/100.

Article Snippet: The used primary antibody was a rabbit polyclonal anti-MSTN antibody against the C-terminal region (300–349 aa) of mouse myostatin (TA343358, OriGene; dilution 1:1000) that presented broad species reactivity, including artiodactyls.

Techniques:

Journal: Frontiers in Genetics

Article Title: Beyond the Big Five: Investigating Myostatin Structure, Polymorphism and Expression in Camelus dromedarius

doi: 10.3389/fgene.2019.00502

Figure Lengend Snippet: Absolute quantification of the myostatin transcript in skeletal muscles. Results of the Digital Droplet PCR analysis for the eight considered dromedary muscles (1, deltoid ; 2, extensor carpi radialis ; 3, coccygeus ; 4, biceps femoris ; 5, peroneus longus ; 6, semitendinosus ; 7, tensor fasciae latae ; 8, braciocephalicus ) and for the two used probes (u, Probe 1; ◆, □ Probe 2) are presented as mean ± SD of the four replicates.

Article Snippet: The used primary antibody was a rabbit polyclonal anti-MSTN antibody against the C-terminal region (300–349 aa) of mouse myostatin (TA343358, OriGene; dilution 1:1000) that presented broad species reactivity, including artiodactyls.

Techniques: Quantitative Proteomics, Muscles

Journal: Frontiers in Genetics

Article Title: Beyond the Big Five: Investigating Myostatin Structure, Polymorphism and Expression in Camelus dromedarius

doi: 10.3389/fgene.2019.00502

Figure Lengend Snippet: Myostatin protein expression. (A) Representative Western Blot of the eight considered dromedary muscles (1, deltoid ; 2, extensor carpi radialis ; 3, coccygeus ; 4, biceps femoris ; 5, peroneus longus ; 6, semitendinosus ; 7, tensor fasciae latae ; 8, brachiocephalicus ) performed using a rabbit polyclonal antibody (1:1000 dilution) that specifically binds the carboxy-terminal domain (Origene, TA343358). (B) Western Blot densitometric analysis of the promyostatin dimer (75 kDa), the promyostatin monomer (40 kDa) and the active C-terminal dimer (25 kDa), respectively. For each lane, optical density (OD) of the considered band is presented as a ratio over the total density of all the proteins transferred on the membrane and stained with Coomassie blue.

Article Snippet: The used primary antibody was a rabbit polyclonal anti-MSTN antibody against the C-terminal region (300–349 aa) of mouse myostatin (TA343358, OriGene; dilution 1:1000) that presented broad species reactivity, including artiodactyls.

Techniques: Expressing, Western Blot, Muscles, Membrane, Staining