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: Endocrinology

Article Title: TGFβ Superfamily Members Mediate Androgen Deprivation Therapy-Induced Obese Frailty in Male Mice.

doi: 10.1210/en.2016-1580

Figure Lengend Snippet: Figure 7. Castration induces myostatin protein levels in skeletal muscle. A, Representative immunoblots of myostatin and -actin (reprobing of the myostatin blot) expression in GAS muscle. B, Quantification of mean myostatin levels in GAS muscles from 4 mice at each time, assessed by 3 independent measurements. C, Representative immunoblot of myostatin and - actin (reprobing of the myostatin blot) expression level in TRI muscle. Lanes of immunoblots marked (C) contain identical control sample for interblot comparison. D, Quantification of mean myostatin levels in TRI muscles from 4 mice at each time, assessed by 3 independent measurements. The values shown in B and D are relative to the sham-castrated (0-wk castrate) animals. Sham-castrated (blue) and castrated groups (red) were compared by one-way ANOVA with Dunnett’s testing. Bars are SEM; *, P .05; **, P .01; ***, P .001 vs sham-castrated group.

Article Snippet: Target Antigen Sequence (if Known) Name of Antibody Manufacturer, Catalog Number, and/or Name of Individual Providing the Antibody Species Raised in; Monoclonal or Polyclonal Dilution Used Myostatin Mouse GDF8 Asn25Ser376 MAb (clone 84214) R&D Systems, MAB788 Monoclonal; rat IgG2B 1:1000 Phospho-Smad2 Smad3S423/5 MAb (clone EP823Y) Abcam, 52903 Monoclonal; rabbit 1:2000 Smad2 Amino terminus hSmad2 L16D3 Cell Signaling, 3103 Monoclonal; mouse 1:2000 Eukaryotic EF2 Complete protein G270 Nastiuk (Ref. 63) Rabbit 1:2000 -Actin -Actin aa 1–14 AC15 Sigma, A5441 Monoclonal; mouse 1:5000 Rat IgG (H L) Rabbit antirat unconjugated Pierce, 31218 Rabbit 1:20 000 Rabbit IgG (H L) Goat antirabbit HRP- conjugated secondary Pierce, 31460 Goat 1:20 000 Mouse IgG (H L) Goat antimouse HRPconjugated secondary Pierce, 31430 Goat 1:20 000 D ow nloaded from https://academ ic.oup.com /endo/article/157/11/4461/2758425 by G ITAM (D eem ed to be U niversity) user on 01 January 2025 tion are an acutely sensitive biological measure of circulating androgen elimination (34, 35).

Techniques: Western Blot, Expressing, Muscles, Control, Comparison

Journal: Endocrinology

Article Title: Reduced Circulating GDF11 Is Unlikely Responsible for Age-Dependent Changes in Mouse Heart, Muscle, and Brain.

doi: 10.1210/en.2015-1628

Figure Lengend Snippet: Figure 1. Circulating myostatin levels decrease in aging mice. A, A myostatin standard curve was generated in the absence or presence of recombinant GDF11. Serum myostatin levels were quantified in 3-month-old wild-type and mstn/ male mice (n 5/group), again in the absence or presence exogenous addition of GDF11 (B, P .05 indicated by different letters) and in three 28 month-old wild-type mice of both sexes (C and D). Significant differences were determined by a regression analysis (C) and by a Student t test (D).

Article Snippet: Assay validation of the R&D System myostatin ELISA (ELISA, catalog No. DGDF80) was performed by running a standard curve in the absence or presence of 2 ng/mL GDF11, also acquired from R&D Systems (catalog No. 1958-GD-010).

Techniques: Generated, Recombinant

Journal: PLoS Genetics

Article Title: Overexpression of Latent TGFβ Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFβ

doi: 10.1371/journal.pgen.1006019

Figure Lengend Snippet: (A) Muscle lysates were fractionated from total lysate (T) into cytosolic (C), light microsome (L) and heavy microsome (H) components. Individual fractions were immunoblotted with an antibody against the active domain of myostatin. There was less myostatin expressed in the soluble fractions of TG+ mdx mice muscle lysates compared to mdx controls. (B) A phylogenetic tree of mouse TGFβ family members derived from protein alignments illustrates the high similarity between these proteins, and explain why antibodies to myostatin cross react to GDF11. (C, D) Mouse LTBP4 and either myc-epitope-tagged myostatin (C) or myc-tagged GDF11 (D) were heterologous expressed in HEK293T cells. The myc-tagged myostatin and GDF11 were the complete prodomains and active domains together. Co-immunoprecipitation was performed on cell lysates by immunoprecipitating with anti-LTBP4 antibody followed by immunoblotting with anti-myc antibody detecting myostatin or GDF11. A co-IP control for each experiment was performed without adding IP antibody (labeled as “No anti”). LTBP4 was found to associate with both myostatin and GDF11 in vitro. The 49 KDa band in the first left lane of panels C and D likely represents endogenous myc.

Article Snippet: Mouse cDNA clones of Mstn (NM_010834), Gdf11 (NM_010272), Tgfβ1 (NM_011577), Tgfβ2 (NM_009367), and Tgfβ3 (NM_009368) were purchased from Origene (catalog numbers MR227629, MR223819, MR227339, MR225633, and MR206441, respectively).

Techniques: Derivative Assay, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Control, Labeling, In Vitro

Journal: PLoS Genetics

Article Title: Overexpression of Latent TGFβ Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFβ

doi: 10.1371/journal.pgen.1006019

Figure Lengend Snippet: RNA sequencing reveals relative abundance of TGFβ family members in muscle.

Article Snippet: Mouse cDNA clones of Mstn (NM_010834), Gdf11 (NM_010272), Tgfβ1 (NM_011577), Tgfβ2 (NM_009367), and Tgfβ3 (NM_009368) were purchased from Origene (catalog numbers MR227629, MR223819, MR227339, MR225633, and MR206441, respectively).

Techniques: RNA Sequencing

Journal: JCI insight

Article Title: Prostate tumor-derived GDF11 accelerates androgen deprivation therapy-induced sarcopenia.

doi: 10.1172/jci.insight.127018

Figure Lengend Snippet: Figure 3. Castration increased active catabol- ic TGF-β family myokine proteins in skeletal muscles of tumor-bearing mice. (A–F) Protein expression in GAS muscle. (A) Representative immunoblots of soluble active myostatin (MSTN) C-terminal dimer and eukaryotic elongation factor 2 (EF2) expression in muscle from sets of 4 mice, castrated for the indicated times or sham castrat- ed. Lanes of immunoblots marked “C” contain identical control sample for interblot compari- son. (B) Quantification of relative MSTN levels for castrated mice (red) or sham-castrated mice (blue), from 3 determinations for each muscle (see Supplemental Figure 5 for additional immunoblots and supplemental materials for full, uncut gels). ELISA-determined protein levels of soluble active activin AA dimer (C), activin BB dimer (D), activin AB dimer (E), and soluble GDF11 (F), in muscle from 4 mice at each time point, measured 3 times each. (G–L) Protein expression in TRI muscle. (G) Representative immunoblot of MSTN and EF2 expression, as in A. (H) Quantification of MSTN levels, as in B. ELISA-determined protein levels of soluble active activin AA dimer (I), activin BB dimer (J), activin AB dimer (K), and soluble GDF11 (L), from 4 mice at each time point, measured 3 times each. Columns are sham-castrated normalized means at each time; bars are SEM. Individual mouse levels are indicated by open circles. *P < 0.05, **P < 0.01, and ***P < 0.001 versus sham-castrated group determined using 1-way ANOVA and Bonferroni’s correction (B and H) or Dunnett’s test (C–F and I–L).

Article Snippet: ELISA kits used were human/mouse/rat activin A ELISA kit (Quantikine DAC00B, R&D Systems, Bio-Techne), mouse activin B ELISA kit (E15932m, Cusabio Biotech Co., Ltd.), mouse activin AB and mouse GDF11 ELISA kits (CEA158Mu and SEC113Mu, respectively, Cloud-Clone Corp.), and total myostatin ELISA kit (Quantikine DGDF80, R&D Systems, Bio-Techne).

Techniques: Muscles, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: JCI insight

Article Title: Prostate tumor-derived GDF11 accelerates androgen deprivation therapy-induced sarcopenia.

doi: 10.1172/jci.insight.127018

Figure Lengend Snippet: Figure 4. TGF-β family myokine ligand blockade reversed castration-induced sarcopenia. (A) Grip strength after castration or sham-castration of mice treated with PBS, ActRIIB-Fc, or anti-GDF11 antibody. (B) Dissected skeletal muscle mass 10 weeks after castra- tion by dissection and weighing. (C) Lean body mass by quantitative NMR (qNMR), as percentage of sham-cas- trated mice. Mean indicated as lines or columns, SEM as bars; n = 3–5/group, indicated by open circles. *P < 0.05 and **P < 0.01 for ActRIIB-Fc treated castrated versus vehicle-treated castrated mice determined using 2-way ANOVA and Tukey’s honestly significant differences (HSD) test. See Supplemental Figure 7 for additional information.

Article Snippet: ELISA kits used were human/mouse/rat activin A ELISA kit (Quantikine DAC00B, R&D Systems, Bio-Techne), mouse activin B ELISA kit (E15932m, Cusabio Biotech Co., Ltd.), mouse activin AB and mouse GDF11 ELISA kits (CEA158Mu and SEC113Mu, respectively, Cloud-Clone Corp.), and total myostatin ELISA kit (Quantikine DGDF80, R&D Systems, Bio-Techne).

Techniques: Dissection

Journal: JCI insight

Article Title: Prostate tumor-derived GDF11 accelerates androgen deprivation therapy-induced sarcopenia.

doi: 10.1172/jci.insight.127018

Figure Lengend Snippet: Figure 5. Castration increased GDF11 and myostatin in tumor and serum. (A–C) GDF11 and myostatin protein expression in tumor. (A) ELISA-determined levels of GDF11 in prostate tumor tissue, from 4 mice at each time, mea- sured 3 times. (B) Representative immunoblots of soluble active myostatin C-terminal dimer (MSTN) and eukaryotic elongation factor 2 (EF2) expression in prostate tumor tissue from sets of 4 mice, castrated for the indicated times or sham castrated. Lanes of immunoblots marked “C” contain identical control sample for interblot comparison. (C) Quantification of MSTN levels in tumor tissue from castrated (red) or sham-castrated (blue) mice, from 3 determi- nations for each tumor (see Supplemental Figure 5 for additional immunoblots and supplemental materials for full, uncut gels). (D–F) GDF11 and myostatin protein expression in serum. (D) ELISA-determined levels of GDF11 in serum, from 4 mice at each time, measured 3 times. (E) Representative immunoblots of MSTN expression in equal quanti- ties of serum protein from the 4 mice in A. (F) Quantification of MSTN levels in serum, as in B. (G–I) Comparison of GDF11 and myostatin protein expression between muscle and tumor. (G) ELISA-determined levels of GDF11 in GAS muscle and prostate tumor from sets of 4 mice, sham castrated (blue) or 2 weeks after castration (red), measured 3 times. (H) Representative immunoblots of MSTN in GAS muscle and prostate tumor from sets of 4 mice, sham-cas- trated or 8 weeks after castration. (I) Quantification of relative levels of MSTN between GAS muscle and prostate tumor. Columns are normalized means; bars are SEM. n = 4/group, indicated by open circles. *P < 0.05, **P < 0.01, and ***P < 0.001 versus sham-castrated group (except G and I, GAS muscle vs. tumor tissue), determined using 1-way ANOVA and Dunnett’s test (A, D, and G) or 1-way ANOVA and Bonferroni’s correction (C, F, and I).

Article Snippet: ELISA kits used were human/mouse/rat activin A ELISA kit (Quantikine DAC00B, R&D Systems, Bio-Techne), mouse activin B ELISA kit (E15932m, Cusabio Biotech Co., Ltd.), mouse activin AB and mouse GDF11 ELISA kits (CEA158Mu and SEC113Mu, respectively, Cloud-Clone Corp.), and total myostatin ELISA kit (Quantikine DGDF80, R&D Systems, Bio-Techne).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Comparison

Journal: JCI insight

Article Title: Prostate tumor-derived GDF11 accelerates androgen deprivation therapy-induced sarcopenia.

doi: 10.1172/jci.insight.127018

Figure Lengend Snippet: Figure 6. Model of ADT-induced catabolic TGF-β family myokine endocrine signaling in PrCa tumor–bearing mice. Graphs of castration-induced changes in myokine concentrations (colored lines) in skeletal muscle and serum of mice without prostate cancer (left side) or in skeletal muscle, serum, and tumor of mice with prostate cancer (right side). Tumor lobes are represented by the green (left lobe) and red (right lobe) circles. In adult tumor-free mice myostatin and the activins increase after castration but prior to strength and muscle mass loss. In PrCa tumor–bearing mice, tumor secretes GDF11 (blue) and later myostatin (red) into serum. Endocrine-derived GDF11 and myostatin increase in muscle prior to strength loss (early myostatin also moves from muscle into serum). The tumor-derived catabolic TGF-β family myokines exacerbate castration-induced sarcopenia in tumor-bearing mice.

Article Snippet: ELISA kits used were human/mouse/rat activin A ELISA kit (Quantikine DAC00B, R&D Systems, Bio-Techne), mouse activin B ELISA kit (E15932m, Cusabio Biotech Co., Ltd.), mouse activin AB and mouse GDF11 ELISA kits (CEA158Mu and SEC113Mu, respectively, Cloud-Clone Corp.), and total myostatin ELISA kit (Quantikine DGDF80, R&D Systems, Bio-Techne).

Techniques: Derivative Assay