Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Determination of efficacy of C2C12 differentiation in conjunction with the exposure to ligand combinations. C2C12s were differentiated for 7 days and treated with combination ligands of GDF11 (G), TMSB4X (T), IL6 (I), and TNF-α (F) at 10 ng/mL for seven additional days. ( A ) Fusion index was calculated from total myotube nuclei vs. total nuclei ( n = 16, mean + SD). ( B ) Multinucleation of C2C12 myotubes were quantified ( n = 11, mean + SD). ( C ) Nuclear density was evaluated from nuclear count per field of 5x microscopy ( n = 4, mean + SD). ( D ) C2C12 exposed to ligand combinations were stained to express nuclear MYOD1 ( n = 6, mean + SD). ( E ) Cells were stained with Ki67, and where similarly quantified based on average total nuclear count ( n = 6, mean + SD). ( F ) ACTN2 and Ki67 immunostaining of control cells. ( G ) Cells exposed to GTF showed decreases in fusion index and myonucleation levels, although no change in nuclear density and Ki67+ expression was detected. ( H ) GTIF supplementation significantly reduced skeletal muscle differentiation parameters fusion index and multinucleation, in addition to decreasing average nuclear density. ( I ) Control C2C12s expressing nuclear MYOD1. ( J ) GTF treatment greatly reduced nuclear fusion and showed limited differentiation capacity while expressing comparable levels of nuclear MYOD1. ( K ) Exposure of C2C12s to GTIF combination significantly inhibited skeletal muscle differentiation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Microscopy, Staining, Immunostaining, Control, Expressing

Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Effect of ligand combination exposure on differentiation of skeletal muscle cells derived from tHFs. Cells were transduced with MYOD1 fragments and induced to express the skeletal muscle phenotype via the induction of doxycycline and SB431542 over a 7-day period. Ligand combinations of GDF11 (G), TMSB4X (T), IL6 (I), and TNF-α (F) at 10 ng/mL were introduced for an additional week, and SB and Dox administration was discontinued. Skeletal muscle cells were fixed and stained on day 14 and characterized by various differentiation and proliferation parameters from 5× microscopy. ( A ) Fusion index of tHFs was evaluated by determining the ratio of myotube nuclei vs total nuclear count ( n = 16, mean + SD). ( B ) Cellular multinucleation was quantified to assess tHF development of differentiation ( n = 22, mean + SD). ( C ) Nuclear density was similarly assessed by quantifying nuclear count per field ( n = 4, mean + SD). ( D ) Nuclear MYOD1 was quantified ( n = 6, mean + SD). ( E ) Ki67 nuclei were also assessed with a nuclear count ( n = 6, mean + SD). ( F ) Control tHF myotubes were immunostained with ACTN2 and Ki67. ( G ) IL6 and TNF-α combination demonstrated significant decrease in differentiation parameters fusion index, multinucleation, myotube length, and diameter , although Ki67+ expression had increased. ( H ) Exposure of tHFs to combined GDF11, TMSB4X, IL6, and TNF-α showed similar results, however nuclear Ki67 expression was unchanged. ( I ) Untreated tHFs with ACTN2 and MYOD1 nuclear stains. ( J ) Cells treated with IF showed a decrease in MYOD1 nuclear expression. ( K ) Additionally, GDF11, TMSB4X, and IL6 exposure yielded similar results with respect to MYOD1+. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Derivative Assay, Transduction, Staining, Microscopy, Control, Expressing

Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Skeletal muscle tissues were engineered from a composite fibrin/Matrigel hydrogel mixture with mouse skeletal myoblasts C2C12s, and subject to 10 ng/mL biological ligands. C2C12s were encapsulated and differentiated in a fibrin-based hybrid hydrogel over a 7-day period, 10 ng/mL biological ligands GDF11, TMSB4X, IL6 or TNF-α were administered after a week of tissue plating. ( A ) Immunohistochemical staining of C2C12 skeletal muscle constructs with ACTN2 and DAPI, demonstrated high cellular density. ( B ) Skeletal myotubes increased compactness and alignment towards central pillar regions where tensile force is maximal ( C ) Structural organization of C2C12s at pillar regions appeared disrupted due to gel contraction. ( D ) Cross-striated, multinucleated skeletal muscle form condensed tissues as demonstrated with high magnification 60× confocal microscopy. ( E ) Myotube diameter (µm) was not affected by one-week exposure to 10 ng/mL ligands. ( n > 32, mean + SD). ( F ) Nuclear density of skeletal muscle C2C12s within tissue were not impacted with ligand administration. ( n = 6, mean + SD).

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Immunohistochemical staining, Staining, Construct, Confocal Microscopy

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: Relative p‐SMAD2/3 (left) and p‐SMAD1/5/8 (right) response, evaluated by AlphaLISA signal, of HEK293T cells after 1‐h exposure to recombinant myostatin (Mstn), activin A, GDF11, TGFβ, and BMP2. Relative p‐SMAD2/3 response of C2C12 myoblasts (left) and myotubes (right) after 1‐h exposure to Mstn, activin A, GDF11, and TGFβ. Phosphorylation of SMAD2 and SMAD3 in differentiated C2C12 myotubes following stimulation with recombinant Mstn or GDF11 for 30 and 60 min, as detected by immunoblotting. Equal loading is verified by Ponceau Red staining. EC 50 values (in nM) for p‐SMAD2/3 and p‐SMAD1/5/8 responses of HEK293T, C2C12 myoblasts, and C2C12 myotubes to the above listed ligands, as well as p‐SMAD1/5/8 response to BMP4, BMP6, and BMP7. Data information: Values are displayed as mean ± SEM; n = 4 for all data points.

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Recombinant, Phospho-proteomics, Western Blot, Staining

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: A, B Significant shifts in the response of HEK293T cells to Mstn, GDF11, and activin A are observed with the addition of ActRIIB antibody (anti‐ActRIIB), when 0 or 100 nM anti‐ActRIIB was applied to HEK293T cells overnight prior to ligand addition and AlphaLISA evaluation. Note that the 0 nM antibody treatment panel is the un‐normalized form of Fig A left. EC 50 values (in nM) are listed for the antibody data in (B). C p‐SMAD1/5/8 response of C2C12 myoblasts (left) and myotubes (right) in response to TGFβ family member ligands. All cells were stimulated with ligand 1 h prior to lysis and evaluation by AlphaLISA. Data Information: Values are displayed as mean ± SEM; n = 4 for all data points.

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Lysis

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: A, B C2C12 myoblasts were differentiated for seven days, then control ( n = 5), myostatin (Mstn; n = 4), growth differentiation factor 11 (GDF11; n = 5), or transforming growth factor β (TGFβ; n = 3) supplemented media (50 ng/ml) was added for 3 days (depicted in A) before PFA fixation and staining with myosin heavy chain (MHC) to evaluate myotube diameter (B). The scale bars represent 50 μm. C, D Myotube diameter data ( n = 200–400 myotubes from 3 to 5 different trials) are depicted as a histogram of diameter distribution (C) and as mean diameter (mean ± SEM; D) by treatment. E Myotube nuclear content was quantified as DAPI‐positive nuclei per μm of myotube length for control, Mstn, and GDF11 treatment groups. F Phosphorylation of SMAD3 in control, Mstn, and GDF11 treatment groups, as detected by immunoblotting. Equal loading is verified by Ponceau Red staining. Data information: Data are depicted as histogram of distribution (C) or mean ± SEM (D, E). Statistical analysis was performed using one‐way ANOVA analysis with Tukey's HSD post hoc test (non‐connecting letters indicate P < 0.05 between groups) and effect size presented as eta‐squared (η 2 ).

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Control, Staining, Phospho-proteomics, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: A Twelve‐week‐old male C57BL/6 mice ( n = 3) were injected i.p. with PBS (control) or 1 × 10 12 gc of a liver‐specific GDF11 packaged in AAV2/8 (AAV8.GDF11), and were euthanized seven days after treatment. B–D Immunoblotting of IgG‐reduced samples (see Materials and Methods) reveals an increase in GDF11 content in AAV8.GDF11‐treated liver (B), serum (C), and quadriceps (D), as detected by the R&D Systems GDF11 mouse mAb under reducing (50 mM DTT) conditions. The identifiable bands of full‐length GDF11 and monomeric GDF11 in the immunoblots are labeled, while an ambiguous 25 kDa band is marked with a star (*). GAPDH immunoblotting is shown to demonstrate equal loading among lanes. E, F Seven days after, AAV8.GDF11 treatment resulted in substantial losses in body weight (E), and muscle mass of the soleus (Sol), extensor digitorum longus (EDL), tibialis anterior (TA), gastrocnemius (Gas), quadriceps (Quad), and heart (F). Values depicted are mean ± SEM. Statistical analysis was performed using two‐tailed Student's t ‐test with effect size presented as Cohen's d ( d ).

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Injection, Control, Western Blot, Labeling, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: A–C Twelve‐week‐old C57BL/6 male mice were injected i.p. with PBS, a liver‐specific myostatin (Mstn) construct packaged into AAV2/8 (AAV8.Mstn), or a liver‐specific GDF11 construct packaged into AAV2/8 (AAV8.GDF11; n = 3). Verification of GDF11 and Mstn overexpression in the liver (A), serum (B), and quadriceps (C) of treated mice using clone 743833 anti‐GDF11 (R&D Systems #MAB19581) and REGN459 anti‐Mstn (Regeneron Pharmaceuticals) mouse monoclonal antibodies using samples pre‐incubated with protein A/G‐coated agarose beads, to reduce endogenous IgGs, and prepared in reducing conditions. The star (*) represents a 25 kDa band that is specifically prominent in AAV8.GDF11‐treated samples, however, is detected by anti‐mouse IgG secondary antibodies. Note that these are the full immunoblot images for those found in Fig B–D. D Immunoblotting for GDF11 (using R&D #MAB19581) with reduced (50 mM DTT) and non‐reduced (no DTT) forms of recombinant GDF11 and Mstn, and liver samples from control, AAV8.Mstn, and AAV8.GDF11 mice subjected to IgG depletion with both protein A/G (targets IgG heavy chains) and protein L (targets IgG light chains)‐coated agarose beads. The differential detection of anti‐mouse IgG immunoreactive bands between AAV8.GDF11‐treated samples and the other groups indicates that GDF11 modifies anti‐mouse IgG immunoreactive species which do not appear to be depletable by incubation with protein A/G or L. The non‐reduced AAV8.GDF11 sample (lane 14) demonstrates both 25 and 12.5 kDa bands immunoreactive with anti‐mouse IgG secondary antibodies, as well. The star (*) represents the ambiguous 25 kDa band mentioned above.

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Injection, Construct, Over Expression, Bioprocessing, Incubation, Western Blot, Recombinant, Control

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: A, B Wheat germ agglutinin (WGA; Texas Red‐conjugated) stained sections of the tibialis anterior (TA; A) and left ventricle (LV; B) from PBS (control) and liver‐specific GDF11 (AAV8.GDF11)‐treated mice ( n = 3) were evaluated for minimum Feret diameter of the individual myocytes ( n = 500–600 cells/group). The scale bars represent 100 μm. Myocyte size data are presented as a histogram of minimum Feret diameter distribution. Statistical analysis was performed using two‐tailed Student's t ‐test with effect size presented as Cohen's d ( d ).

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Staining, Control, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: Four‐week‐old C57BL/6 male mice were injected IP with PBS (control; n = 6) or 1 × 10 12 gc of AAV2/8 packaged liver‐specific constructs of full‐length Mstn (AAV8.Mstn; n = 7), Mstn D76A propeptide (AAV8.dnMstn; n = 3), or GDF11 D120A propeptide (AAV8.dnGDF11; n = 4) and euthanized at 16 weeks of age (84 day treatments). Body weights and muscle masses of 12‐week‐old Mstn WT/WT ( n = 6), Mstn WT/KO ( n = 6), and Mstn KO/KO ( n = 4) mice congenic on a C57BL/6 background. Data Information: Values are presented as mean ± SEM. Statistical analysis performed using one‐way ANOVA analysis with Tukey's HSD post hoc test (non‐connecting letters indicate P < 0.05 between groups) and effect size presented as eta‐squared (η 2 ).

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Injection, Control, Construct

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: Immunoblotting data from quadriceps of PBS (control; n = 3) and liver‐specific GDF11 (AAV8.GDF11; n = 3)‐treated male C57BL/6 mice for phosphorylated and total forms of SMAD3, Akt, and p38 MAPK. Loading is normalized by GAPDH content and quantified relative to control values. Immunoblotting data for phosphorylated and total forms of SMAD3, p38 MAPK, and p42/p44 ERK in the hearts of control and AAV8.GDF11‐treated male C57BL/6 mice. Loading is normalized by GAPDH content and quantified relative to control values. Phosphorylation status of SMAD3 in the hearts of control ( n = 3) and AAV8.GDF11‐treated male C57BL/6 mice at 3 days ( n = 4) and 5 days ( n = 4) following injection. Loading is normalized by Ponceau Red staining and quantified relative to control values. Gene expression of Fbxo32 (MAFbx gene), Trim63 (MuRF1 gene), and Fbxo30 (MUSA‐1 gene) in the quadriceps (left) and heart (right) of control ( n = 3) and AAV8.GDF11‐treated male C57BL/6 mice at 3 days ( n = 4) and 5 days ( n = 4) following injection. Relative gene expression values were calculated by the ΔΔC t method using Gapdh as the reference gene. Data information: Values depicted are mean ± SEM. In (A, B), statistical analysis was performed using two‐tailed Student's t ‐test with effect size presented as Cohen's d ( d ). In (C, D), statistical analysis was performed using one‐way ANOVA analysis with Tukey's HSD post hoc test (non‐connecting letters indicate P < 0.05 between groups) and effect size presented as eta‐squared (η 2 ).

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Western Blot, Control, Phospho-proteomics, Injection, Staining, Gene Expression, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: A, B Quadriceps (A) and heart (B) lysates from mice described in Figs and were immunoblotted for phosphorylated and total content of SMAD3, SMAD1/5/8, Akt, p38 MAPK, ERK1/2, total SMAD4, and total NOX4. GAPDH content was used as a loading control and normalization standard. Note that the cropped version of many of these images is found in Fig A and B. C Phosphorylation of TAK1 is not different between control and AAV8.GDF11‐treated hearts or quadriceps.

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Control, Phospho-proteomics

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: Eleven‐week‐old C57BL/6 male mice received i.p. injections of PBS (control; n = 4), 1 × 10 11 gc of AAV8.GDF11 (mid dose; n = 3), or 5 × 10 10 gc of AAV8.GDF11 (low dose; n = 3), and their body weights were monitored every other day until the 1 × 10 11 gc AAV8.GDF11 group required euthanasia on day 10. Immunoblotting of serum samples for GDF11 demonstrates expression levels of 0.5–1.6 ng/μl and 0.26–0.43 ng/μl for the mid‐ and low‐dose groups, respectively. Change in mouse body weights (Bwt) across the 10‐day study by the AAV8.GDF11 treatment groups, including terminal values for the previous 7‐day cohort treated with 1 × 10 12 gc of AAV8.GDF11 (high dose). Mass of the quadriceps, gastrocnemius, and heart of the 10‐day study mice ( n = 12 for control mice by the inclusion of untreated age‐matched mice, resulting in a larger, homogenous data set). Liver and kidney mass of 7‐week‐old C57BL/6 male mice were treated with PBS (control; n = 5), AAV8.GDF11 low dose ( n = 4), or AAV8.Mstn high dose ( n = 5) for 16 days (see Fig B). The mean values for 7‐week‐old mice from this colony ( n = 5) are indicated by the dotted line to show starting masses. Immunoblotting comparison of monomeric GDF11 and Mstn levels in quadriceps, heart, and kidney demonstrate that differential effects are not due to differential accumulation of the ligands in tissue. Data information: Values are displayed as mean ± SEM. Statistical analysis performed using one‐way ANOVA analysis with Tukey's HSD post hoc test (non‐connecting letters indicate P < 0.05 between groups) and effect size presented as eta‐squared (η 2 ). Note that “b” on Day 4 of panel (C) refers to the 2 overlapping groups.

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Control, Western Blot, Expressing, Comparison

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: A Nearly equivalent serum exposure of myostatin (Mstn) and GDF11 can be obtained by treatment with 2 × 10 12 gc of AAV8.Mstn (high dose) or 5 × 10 10 gc of AAV8.GDF11 (low dose) 10 days following injection. B, C Seven‐week‐old C57BL/6 male mice were treated with PBS (control; n = 5), AAV8.GDF11 low dose ( n = 5), or AAV8.Mstn high dose ( n = 5) and monitored for 16 days until experiment was terminated due to the death of an AAV8.GDF11‐treated mouse to severe cachexia (depicted in B). The change in body weight (Bwt) in these groups across the 16 days is displayed in (C). D–G Morphological measurements of surviving mice (thus n = 4 for AAV8.GDF11 group) collected at tissue harvest, including Bwt (D), muscle mass of soleus and extensor digitorum longus (EDL), tibialis anterior (TA), gastrocnemius (Gastroc), quadriceps (Quad), and heart (E–F). Heart mass normalized to both Bwt (in g) and tibia length (TL; in mm; G). Mean values for 7‐week‐old mice from this colony ( n = 5) are indicated by the dotted lines to show starting masses. Data information: Values are displayed as mean ± SEM. Statistical analysis performed using one‐way ANOVA analysis with Tukey's HSD post hoc test (non‐connecting letters indicate P < 0.05 between groups) and effect size presented as eta‐squared (η 2 ) for ANOVA analyses. Note that “a” on Day 2 of panel (C) refers to the 2 overlapping groups.

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Injection, Control

Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: A, B Relative muscle content of the activin type IIB receptor (ActRIIB) in quadriceps ( n = 6) and hearts ( n = 4) from multiple treatment groups, as determined by immunoblotting (normalized to Ponceau‐visualized loading). C–F Gene expression of Acvr1b (ALK4 gene; C), Tgfbr1 (ALK5 gene; D and E), and Tgfb1 (F) in quadriceps and heart of untreated 7‐week‐old C57BL/6 mice ( n = 3), as measured by real‐time PCR. Relative gene expression values were calculated by the ΔΔC t method using Gapdh (C, D and F) or Acvr1b (E) as reference genes. G Cardiac gene expression of Tgfb1 in control ( n = 3), day 3 ( n = 4), and day 5 ( n = 4) high‐dose (1 × 10 12 gc) AAV8.GDF11‐treated C57BL/6 mice. Relative gene expression values were calculated by the ΔΔC t method using Gapdh as the reference gene. Data information: Values are displayed as mean ± SEM. In (B–F), statistical analysis performed using two‐tailed Student's t ‐test, with effect size presented as Cohen's d ( d ). In (G), statistical analysis performed using one‐way ANOVA with Tukey's HSD post hoc test (non‐connecting letters indicate P < 0.05 between groups) and effect size presented as eta‐squared (η 2 ).

Article Snippet: Primary antibodies used for this study include the following: GDF11 (R&D Systems #MAB19581; Minneapolis, MN), Mstn (C‐terminal; kind gift from Regeneron Pharmaceuticals), p‐SMAD2 (S465/467; Cell Signaling #3108), SMAD2 (Cell Signaling #5339), p‐SMAD3 (S423/425; Cell Signaling #9520), SMAD3 (Cell Signaling #9523), p‐SMAD1/5/8 (S463/465; Cell Signaling #9511), SMAD5 (Cell Signaling #12534), SMAD4 (Cell Signaling #9515), p‐Akt (S473; Cell Signaling #9271), Akt (Cell Signaling #9272), p‐p38 MAPK (T180/Y182; Cell Signaling #9211), p38 MAPK (Cell Signaling #9212), p‐ERK1/2 (T204/Y204; Cell Signaling #9101), ERK1/2 (Cell Signaling #9102), NOX4 (Abcam #ab133303; Cambridge, MA), p‐TAK1 (T184/187; Cell Signaling #4531), TAK1 (Cell Signaling #4505), ActRIIB (Sigma‐Aldrich #A0457), and Mstn/GDF11 (Abcam #ab124721).

Techniques: Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Control, Two Tailed Test

Journal: Physiological Reports

Article Title: Lifelong exercise, but not short‐term high‐intensity interval training, increases GDF 11, a marker of successful aging: a preliminary investigation

doi: 10.14814/phy2.13343

Figure Lengend Snippet: Effect of HIIT on growth factor family members in SED ( N = 13) and LEX ( N = 11) individuals. Concentration of (A) total myostatin, (B) free myostatin, (C) follistatin and (D) GDF 11 pre‐ and post‐ HIIT . Note (D) is expressed on a logarithmic y axis. HIIT, high‐intensity interval training; LEX, lifelong exercising; SED, sedentary; GDF11, growth and differentiation factor 11.

Article Snippet: Concentrations of serum follistatin (DFN00; R&D Systems, Abingdon, UK) and serum GDF11 (DY1958; R&D Systems) were quantified by ELISA, per manufacturer's instructions.

Techniques: Concentration Assay

Journal: Physiological Reports

Article Title: Lifelong exercise, but not short‐term high‐intensity interval training, increases GDF 11, a marker of successful aging: a preliminary investigation

doi: 10.14814/phy2.13343

Figure Lengend Snippet: Correlations between myostatin‐interacting factors and peak power output (W). (A) Peak power output (W) by group ( SED , LEX ). Horizontal solid lines indicate group mean. (B) GDF 11 (pg/mL) as a function of peak power output (W). (C) GDF 11 (pg/mL) as a function of peak power output (W/kg FFM). Dashed line indicates 95% confidence intervals. Closed circles are SED ( N = 13), open squares are LEX ( N = 11). LEX, lifelong exercising; SED, sedentary; GDF11, growth and differentiation factor 11.

Article Snippet: Concentrations of serum follistatin (DFN00; R&D Systems, Abingdon, UK) and serum GDF11 (DY1958; R&D Systems) were quantified by ELISA, per manufacturer's instructions.

Techniques:

Journal: Bone Research

Article Title: Relationship of serum GDF11 levels with bone mineral density and bone turnover markers in postmenopausal Chinese women

doi: 10.1038/boneres.2016.12

Figure Lengend Snippet: Subject characteristics and bone and biochemical parameters of 169 postmenopausal women

Article Snippet: High bind 96-well multi-array plates were coated with 50 μL per well of 2 mg·mL −1 mouse anti-human GDF11 (R&D Systems, clone 743833) in phosphate-buffered saline (PBS) overnight at 4 °C.

Techniques:

Journal: Bone Research

Article Title: Relationship of serum GDF11 levels with bone mineral density and bone turnover markers in postmenopausal Chinese women

doi: 10.1038/boneres.2016.12

Figure Lengend Snippet: GDF11 serum levels increase with aging. GDF11 positively correlates with age ( r =0.601, P <0.01). Scatter plot of serum GDF11 concentration versus age.

Article Snippet: High bind 96-well multi-array plates were coated with 50 μL per well of 2 mg·mL −1 mouse anti-human GDF11 (R&D Systems, clone 743833) in phosphate-buffered saline (PBS) overnight at 4 °C.

Techniques: Concentration Assay

Journal: Bone Research

Article Title: Relationship of serum GDF11 levels with bone mineral density and bone turnover markers in postmenopausal Chinese women

doi: 10.1038/boneres.2016.12

Figure Lengend Snippet: Correlation coefficients of serum GDF11 and BMD

Article Snippet: High bind 96-well multi-array plates were coated with 50 μL per well of 2 mg·mL −1 mouse anti-human GDF11 (R&D Systems, clone 743833) in phosphate-buffered saline (PBS) overnight at 4 °C.

Techniques:

Journal: Bone Research

Article Title: Relationship of serum GDF11 levels with bone mineral density and bone turnover markers in postmenopausal Chinese women

doi: 10.1038/boneres.2016.12

Figure Lengend Snippet: Parameters identified as significant and independent predictors of BMD

Article Snippet: High bind 96-well multi-array plates were coated with 50 μL per well of 2 mg·mL −1 mouse anti-human GDF11 (R&D Systems, clone 743833) in phosphate-buffered saline (PBS) overnight at 4 °C.

Techniques:

Journal: Bone Research

Article Title: Relationship of serum GDF11 levels with bone mineral density and bone turnover markers in postmenopausal Chinese women

doi: 10.1038/boneres.2016.12

Figure Lengend Snippet: Correlation of serum GDF11 levels with bone turnover markers

Article Snippet: High bind 96-well multi-array plates were coated with 50 μL per well of 2 mg·mL −1 mouse anti-human GDF11 (R&D Systems, clone 743833) in phosphate-buffered saline (PBS) overnight at 4 °C.

Techniques: