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: Frontiers in endocrinology

Article Title: Acute endurance exercise modulates growth differentiation factor 11 in cerebrospinal fluid of healthy young adults.

doi: 10.3389/fendo.2023.1137048

Figure Lengend Snippet: FIGURE 1 An acute bout of endurance exercise (90 min run) modified the levels of GDF11 in cerebrospinal fluid but not in circulation of healthy trained volunteers. GDF11 levels were assessed by immunoblotting, using three different primary antibodies: (A) GDF11-specific N-terminus antibody (CSF, n=12/12, B/R, p=0.046); (B) GDF11-specific C-terminus antibody (CSF, n=13/13, B/R, p=0.008); (C) GDF11/8 antibody (CSF, n=12/12, B/R, p=0.065); (D) GDF11-specific N-terminus antibody (serum, n= 14/14/14, B/0/R); (E) GDF11-C-terminus antibody (plasma, n=13/13/13, B/0/R); (F) GDF11- specific N-terminus antibody (plasma, n=14/14/13, B/0/R); Due to the lack of space, molecular weight ladders are not visible in Figures 1D–F and therefore, the molecular weights of GDF11 are in this case approximate, based on our validation studies of antibodies. B, Basal, before run; R, after run; A.U., signal intensity; GDF, Growth Differentiation Factor. Immunoblotting of all samples was performed under identical conditions. Statistical differences were analysed using paired Student’s t-test. *p<0.05, **p<0.01, #p<0.1 (A–C).

Article Snippet: To corroborate the findings, GDF11 C-terminus specific monoclonal antibody was used (recombinant human GDF11 aa299-407, 1:1000, R&D systems, MAB19581, USA) (sample set B, n=13; overlap between A & B sample sets: n=7, Supplementary Figure 1B).

Techniques: Western Blot, Clinical Proteomics, Molecular Weight, Biomarker Discovery

Journal: Frontiers in endocrinology

Article Title: Acute endurance exercise modulates growth differentiation factor 11 in cerebrospinal fluid of healthy young adults.

doi: 10.3389/fendo.2023.1137048

Figure Lengend Snippet: FIGURE 2 Running-induced decrease of blood-brain barrier permeability (CSF/serum albumin ratio). n=17. (albumin values were not measured in CSF of one individual, and two individuals had CSF available only after the run) (A); its association with CSF/serum GDF11 ratio (B); and CSF/serum GDF11/8 ratio (C). CSF, Cerebrospinal Fluid; GDF, Growth Differentiation Factor. ***p<0.001; Open circles - before run; Full circles - after run.

Article Snippet: To corroborate the findings, GDF11 C-terminus specific monoclonal antibody was used (recombinant human GDF11 aa299-407, 1:1000, R&D systems, MAB19581, USA) (sample set B, n=13; overlap between A & B sample sets: n=7, Supplementary Figure 1B).

Techniques: Permeability

Journal: Frontiers in endocrinology

Article Title: Acute endurance exercise modulates growth differentiation factor 11 in cerebrospinal fluid of healthy young adults.

doi: 10.3389/fendo.2023.1137048

Figure Lengend Snippet: FIGURE 3 Heat map depicting associations between levels of GDF11 and clinical parameters, as well as their running-induced changes. Data were analyzed using GDF11-specific N-terminus antibody (paired serum samples n=14; paired CSF samples n=12, unpaired CSF samples available only after run n=2). BMI, body mass index; CSF, cerebrospinal fluid; GDF, Growth Differentiation Factor; HRmax, maximal heart rate; S-CK, serum creatinkinase; R, correlation coefficient; VO2max, maximal aerobic capacity. Coefficients of significant correlations (p<0.05) or trends (p<0.1) are presented in numbers.

Article Snippet: To corroborate the findings, GDF11 C-terminus specific monoclonal antibody was used (recombinant human GDF11 aa299-407, 1:1000, R&D systems, MAB19581, USA) (sample set B, n=13; overlap between A & B sample sets: n=7, Supplementary Figure 1B).

Techniques:

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