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

Journal: Molecular neurobiology

Article Title: Endoplasmic Reticulum Stress Induces Myostatin High Molecular Weight Aggregates and Impairs Mature Myostatin Secretion.

doi: 10.1007/s12035-018-0997-9

Figure Lengend Snippet: Fig. 1 Presence of anti-MstnPP and anti-APP immunoreactivity in sIBM myofibers. a Hematoxylin and eosin staining of representative sIBM muscle biopsy, showing variation in muscle fiber size as well degenerative changes, in particular typical rimmed vacuoles (arrowhead) and basophilic sarcoplasmic inclusions. b Rimmed vacuoles (arrowhead) stained with modified Gomori trichrome stain. c Congo Red fluorescence of myofibers demonstrates sarcoplasmic amyloid deposits, in particular adjacent to vacuoles. One optical plane is shown in all images. d, e Representative transverse serial sections of skeletal muscle from (d) sIBM patients, (e) age-matched controls and neurogenic patients. Sections were stained with anti-APP 6E10 directed against the amyloidogenic Aβ region and anti-MstnPP 6H12 directed against the propeptide region of MstnPP (anti-Mstn-N). Nuclei were stained with Hoechst. Scale bars 10 μm

Article Snippet: The human MstnPP cDNA (Origene) was used for PCR and the open reading frame was cloned into the lentiviral vector pRRLsin.PPT.CMV.Wpre [41].

Techniques: Staining, Modification, Fluorescence

Journal: Molecular neurobiology

Article Title: Endoplasmic Reticulum Stress Induces Myostatin High Molecular Weight Aggregates and Impairs Mature Myostatin Secretion.

doi: 10.1007/s12035-018-0997-9

Figure Lengend Snippet: Fig. 2 APP and MstnPP metabolites co-localize with ER marker Calreticulin around rimmed vacuoles in sIBM biopsies. Confocal microscopy analysis of representative sIBM patient muscle biopsies co-stained for either (a) APP (antibodies 6E10 and 22C11, green) or (b) MstnPP (6H12, green) and Calreticulin (red). I and II represent sets of consecutive sections. Antibodies directed against the aminoterminus (22C11) or the Aβ peptide region (6E10) both stain inclusions and boundaries of degenerative vacuoles. Antibody 6H12 directed against Mstn-N reacts with propeptide and pro-Mstn. Prominent staining is found around rimmed vacuoles and co-localizes with Calreticulin. Scale bar 10 μm

Article Snippet: The human MstnPP cDNA (Origene) was used for PCR and the open reading frame was cloned into the lentiviral vector pRRLsin.PPT.CMV.Wpre [41].

Techniques: Marker, Confocal Microscopy, Staining

Journal: Molecular neurobiology

Article Title: Endoplasmic Reticulum Stress Induces Myostatin High Molecular Weight Aggregates and Impairs Mature Myostatin Secretion.

doi: 10.1007/s12035-018-0997-9

Figure Lengend Snippet: Fig. 5 Increased MstnPP metabolites cause ER stress and form SDS- insoluble aggregates. a Glutaraldehyde crosslinking of proteins. Lysates were incubated with 0.005% glutaraldehyde and proteins were separated by SDS-PAGE. Pro-Mstn dimers with intact disulfide bonds (dots), high molecular weight species in the stacking gel (white arrowhead), pro-Mstn (black arrowhead) and propeptide (star) are indicated. b SDD-AGE analysis reveals presence of aggregated MstnPP metabolites but not APP. Cell lysates were mixed with 2% SDS containing sample buffer and proteins were separated by agarose gel electrophoresis. Arrowhead indicates monomeric, parenthesis indicates polymeric proteins. Please note that molecular weight cannot be determined by SDD-AGE [44]. c Western blot analysis of BIP expression in WT and transgenic cell lines. Blots were probed with anti-APP (6E10), anti-Mstn-N (6H12), anti-GFP and anti-BIP antibody. Actin served as a loading control. WT: untransduced CCL 136 cells. d Fold change in BIP expression in CCL 136 cells overexpressing MstnPP, APP or EGFP compared to WT controls. Bars represent mean values ± SD (n = 4). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparison test. Significant changes are indicated by asterisks ***p < 0.001, ns = not significant. e Percentage of cells in transduced bulk populations overexpressing the transgenes as determined by automated confocal microscopy. Fixed cells were stained for APP (antibody 6E10) or MstnPP (6H12). Per cell line, eight wells were plated. Wildtype cells served as negative controls. At least 5000 cells per cell population were counted

Article Snippet: The human MstnPP cDNA (Origene) was used for PCR and the open reading frame was cloned into the lentiviral vector pRRLsin.PPT.CMV.Wpre [41].

Techniques: Incubation, SDS Page, High Molecular Weight, Agarose Gel Electrophoresis, Molecular Weight, Western Blot, Expressing, Transgenic Assay, Control, Comparison, Confocal Microscopy, Staining