Journal: Skeletal Muscle

Article Title: A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice

doi: 10.1186/s13395-019-0197-y

Figure Lengend Snippet: GDF11PRO-Fc associates with GDF11 and MSTN. Protein-protein interactions between GDF11PRO-Fc or MPRO-Fc and rGDF11, rMSTN, or rActivin A were determined by a pull-down assay. GDF11PRO-Fc or MPRO-Fc was incubated with rGDF11, rMSTN, or rActivin A for 1 h at 4 °C. Fc-fused protein complexes were separated on a protein A/G-coated agarose resin and eluates were run on a 12% SDS-PAGE gel under reducing conditions and probed by western blot. Input control was 5% of the input material. WB western blot

Article Snippet: To assess complex formation, rGDF11 (1958-GD-010; R&D Systems; Minneapolis, MN), recombinant MSTN (rMSTN; 788-G8–010; R&D Systems; Minneapolis, MN) or recombinant activin A (rActivinA; 338-AC-010; R&D Systems; Minneapolis, MN) were added to cell lysates to a final concentration of 100–500 ng/ml.

Techniques: Protein-Protein interactions, Pull Down Assay, Incubation, SDS Page, Western Blot, Control

Journal: Skeletal Muscle

Article Title: A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice

doi: 10.1186/s13395-019-0197-y

Figure Lengend Snippet: GDF11PRO-Fc blocks GDF11/MSTN-induced myotube atrophy in C2C12 cells. a Schematic detailing experimental timeline in C2C12 myotubes. AAV6-EGFP or AAV6-GDF11PRO-Fc was added to C2C12 myotubes at a MOI of 10 on day 5 post-differentiation and 100 ng/ml rGDF11 or rMSTN was added on day 7. Myotubes were stained and analyzed on day 10. b EGFP expression was evident at 48–72 h in C2C12 myotubes treated with AAV6-EGFP (MOI 10 ). Scale bars represents 50 μm. c Vector genome copy number per diploid genome in C2C12 myotubes 72 h after addition of AAV6-EGFP or AAV6-GDF11PRO-Fc (MOI 10 ). d Representative immunofluorescence images of C2C12 myotubes. C2C12 myotube membranes were visualized by staining with an anti-dystrophin antibody (red). Nuclei were stained with DAPI (blue). Inset shows a zoomed-in region. Scale bars represent 50 μm (main panel) and 25 μm (panel inset). e The fraction of nuclei incorporated into myotubes (differentiation index) was calculated and presented as a percentage of control. f Average myotube diameter relative to control and ( g ) distribution of diameter measurements. For myotube diameter measurements, each myotube was measured at three points along the length of the myotube and averaged. h Number of nuclei incorporated per myotube. A minimum of 50 myotubes were analyzed per experimental condition. i pSMAD2/3 relative to tSMAD2/3 was assessed by western blot. Equal protein loading was verified by Ponceau S staining and GAPDH was used as a loading control. Data represents results from three separate experiments. All error bars represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. not significant; compared to AAV6-EGFP-treated control. † p < 0.05; †† p < 0.01; ††† p < 0.001; compared to AAV6-EGFP + ligand-treated. pSMAD2/3: phosphorylated SMAD2/3; tSMAD2/3: total SMAD2/3

Article Snippet: To assess complex formation, rGDF11 (1958-GD-010; R&D Systems; Minneapolis, MN), recombinant MSTN (rMSTN; 788-G8–010; R&D Systems; Minneapolis, MN) or recombinant activin A (rActivinA; 338-AC-010; R&D Systems; Minneapolis, MN) were added to cell lysates to a final concentration of 100–500 ng/ml.

Techniques: Staining, Expressing, Plasmid Preparation, Immunofluorescence, Control, Western Blot

Journal: Molecular medicine reports

Article Title: Growth differentiation factor 11 is involved in isoproterenol‑induced heart failure.

doi: 10.3892/mmr.2019.10077

Figure Lengend Snippet: Figure 3. GDF11 reduces the proliferation and increases LDH release of ISO‑treated H9C2 cells in a dose‑dependent manner. (A) Proliferation of H9C2 cells was measured using a Cell Counting Kit‑8 assay. (B) LDH levels in H9C2 cells. Data are presented as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control group; #P<0.05 vs. ISO group. rGDF11, recombinant growth differentiation factor 11; ISO, isoproterenol; LDH, lactate dehydrogenase.

Article Snippet: H9C2 cells were categorized into three groups based on treatment: i) Control group (cells were cultured in DMEM for 24 h and then treated with saline at 37 ̊C for 24 h); ii) ISO group (cells were cultured in DMEM for 24 h and then treated with 10 μM ISO at 37 ̊C for 24 h); and iii) ISO + rGDF11 group [cells were pre-incubated with different concentrations (0.5, 5 or 50 nM) of rGDF11 protein (cat. no. 1958-GD; R&D Systems, Inc., Minneapolis, MN, USA) (22) at 37 ̊C for 24 h and then treated with 10 μM ISO at 37 ̊C for 24 h].

Techniques: CCK-8 Assay, Control, Recombinant

Journal: Molecular medicine reports

Article Title: Growth differentiation factor 11 is involved in isoproterenol‑induced heart failure.

doi: 10.3892/mmr.2019.10077

Figure Lengend Snippet: Figure 5. GDF11 increases ISO‑induced oxidative stress by upregulating Nox4 in H9C2 cells. (A) Levels of reactive oxygen species in H9C2 cells determined by DHE staining. (B) Concentration of MDA in H9C2 cells. (C) Representative western blot and quantitative analyses of (D) Nox4 and (E) Nox2 expression in H9C2 cells. Data are presented as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control group; #P<0.05 vs. ISO group. DHE, dihydroethidium; rGDF11, recombinant growth differentiation factor 11; ISO, isoproterenol; MDA, malondialdehyde; Nox, nicotinamide adenine dinucleotide phosphate oxidase.

Article Snippet: H9C2 cells were categorized into three groups based on treatment: i) Control group (cells were cultured in DMEM for 24 h and then treated with saline at 37 ̊C for 24 h); ii) ISO group (cells were cultured in DMEM for 24 h and then treated with 10 μM ISO at 37 ̊C for 24 h); and iii) ISO + rGDF11 group [cells were pre-incubated with different concentrations (0.5, 5 or 50 nM) of rGDF11 protein (cat. no. 1958-GD; R&D Systems, Inc., Minneapolis, MN, USA) (22) at 37 ̊C for 24 h and then treated with 10 μM ISO at 37 ̊C for 24 h].

Techniques: Staining, Concentration Assay, Western Blot, Expressing, Control, Recombinant

Journal: Molecular medicine reports

Article Title: Growth differentiation factor 11 is involved in isoproterenol‑induced heart failure.

doi: 10.3892/mmr.2019.10077

Figure Lengend Snippet: Figure 4. GDF11 aggravates ISO‑induced cell damage in H9C2 cells. (A) Proliferation of H9C2 cells was measured using a Cell Counting Kit‑8 assay. (B) Levels of LDH release from H9C2 cells. (C) Representative western blot and (D) quantitative analyses of Bax and Bcl‑2 protein expression in treated H9C2 cells. (E) Caspase‑3 activity in H9C2 cells. (F) Quantitative analysis and (G) representative images of apoptotic H9C2 cells stained with Hoechst 33258. Scale bar, 50 µm. Data are presented as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. control group; #P<0.05 vs. ISO group. Bcl‑2, B‑cell lymphoma 2; Bax, Bcl‑2‑associated X protein; rGDF11, recombinant growth differentiation factor 11; ISO, isoproterenol.

Article Snippet: H9C2 cells were categorized into three groups based on treatment: i) Control group (cells were cultured in DMEM for 24 h and then treated with saline at 37 ̊C for 24 h); ii) ISO group (cells were cultured in DMEM for 24 h and then treated with 10 μM ISO at 37 ̊C for 24 h); and iii) ISO + rGDF11 group [cells were pre-incubated with different concentrations (0.5, 5 or 50 nM) of rGDF11 protein (cat. no. 1958-GD; R&D Systems, Inc., Minneapolis, MN, USA) (22) at 37 ̊C for 24 h and then treated with 10 μM ISO at 37 ̊C for 24 h].

Techniques: CCK-8 Assay, Western Blot, Expressing, Activity Assay, Staining, Control, Recombinant