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: 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

Journal: Molecular cancer research : MCR

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1158/1541-7786.MCR-25-0211

Figure Lengend Snippet: 293T cells co-expressing PCSK5 M452I secrete mature ectopic GDF11 with intermediate efficiency. A, Catalytically active PCSK5 promotes ectopic GDF11 secretion. Cells were lipofected with 3 ng of the indicated PCSK5 allele (or EGFP overexpression control) plus 100 ng of GDF11, and conditioned medium was collected after 24 hours to measure GDF11 release by ELISA. PCSK5 T288P was included as a catalytically dead control ( 28 ). B, GDF11 release by PCSK5 M452I plus wildtype PCSK5 (PCSK5 WT ) is additive. Cells were treated as in ( A ) and compared with 1.5 ng PCSK5 WT plus 1.5 ng PCSK5 M452I . C, Cotransfection with oncogenic HRAS G12V approximates the level of prenylated HRAS (HRAS prenyl ) in MCF10DCIS.com. 293T cells were lipofected with 2 ng of HRAS G12V and 3 ng of the indicated PCSK5 allele (or EGFP overexpression control) and immunoblotted for HRAS with vinculin and p38 used as loading controls. MCF10A-5E cells are a negative control for HRAS overexpression. D, HRAS G12V cotransfection does not alter the relative GDF11 secretion efficiencies of wildtype PCSK5, PCSK5 M452I , and PCSK5 T288P . Cells were treated as in ( C ) and measured for GDF11 release by ELISA. For ( A ), ( B ), and ( D ), GDF11 ELISA results are normalized to the GDF11-only condition [gray dashed in ( A )] and shown as the mean ± SEM of N = 4 biological replicates. Differences among +GDF11 groups were analyzed by multiway ANOVA with PCSK5 genotype as a fixed effect. Significant factors were followed up pairwise by Tukey-Kramer post hoc analysis.

Article Snippet: After 24 hours, conditioned medium was collected, and the concentration of secreted GDF11 was quantified with a GDF11 ELISA (R&D Systems, DY1958).

Techniques: Expressing, Over Expression, Control, Enzyme-linked Immunosorbent Assay, Cotransfection, Negative Control

Journal: Molecular cancer research : MCR

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1158/1541-7786.MCR-25-0211

Figure Lengend Snippet: Inducible reconstitution of PCSK5 alleles in PCSK5 −/− MCF10DCIS.com cells. A, Approach to MCF10DCIS.com engineering. Cells were transduced with a destabilizing domain (DD)-containing Cas9-P2A-Venus ( 61 ) and a single-guide RNA targeting Exon 4 of PCSK5 (sgPCSK5). Transduced cells were treated with 200 nM Shield-1 ( 61 ) to stabilize Cas9-P2A-Venus and 2% matrigel to promote PCSK5 accessibility before sorting single Venus-positive cells into 10 ng/ml GDF11 (to aid recovery upon PCSK5 loss) and screening genomic DNA (gDNA) of expanded clones for knockout. One confirmed PCSK5 −/− clone was then transduced with sgPCSK5-resistant, tetracycline (tet)-regulated, V5-tagged alleles of PCSK5 and selected polyclonally for hygromycin resistance. B, Sequence-confirmed knockout alleles of MCF10DCIS.com clone 3D8. The PCSK5 coding sequence (CDS) is shown with annotations for the signal peptide (SP, purple), proprotein sequence (Pro, green), and peptidase domain (blue) including its catalytic triad (yellow stars). The protospacer adjacent motif (PAM) of sgPCSK5 is just upstream of the first triad amino acid, and deletions (white, Allele 1) or insertions (pink, Allele 2) induce frameshift mutations (gray) removing the first amino acid in the catalytic triad (black outlined stars) and producing premature stop codons (red). C, Quantification of reconstituted PCSK5 alleles by calibrating against recombinant V5-containing Multitag protein at the indicated copies per cell ( 63 , 72 ). Cells were treated with 1 μg/ml doxycycline for 24 hours, and total protein from counted cells was immunoblotted by two-color fluorescence detection for V5 (800 channel) with tubulin and p38 (700 channel) used as loading controls for cells. Copy number estimates are: PCSK5 WT , 136,000 ± 11,000 copies per cell; PCSK5 M452I , 164,000 ± 6,000 copies per cell; PCSK5 T288P , 176,000 ± 15,000 copies per cell ( N = 4 biological replicates).

Article Snippet: After 24 hours, conditioned medium was collected, and the concentration of secreted GDF11 was quantified with a GDF11 ELISA (R&D Systems, DY1958).

Techniques: Transduction, Clone Assay, Knock-Out, Sequencing, Recombinant, Fluorescence

Journal: Molecular cancer research : MCR

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1158/1541-7786.MCR-25-0211

Figure Lengend Snippet: PCSK5 activity promotes rounded multi-cell organization in 3D matrigel cultures of MCF10DCIS.com. A, Spheroid growth rates for the indicated PCSK5 addback lines estimated by nonlinear least-squares regression of cross-sectional area ( 50 ) at 4, 8, and 12 days from N = 8 biological replicates (gray dashed). B and C , Reduced multi-cell circularity of MCF10DCIS.com upon loss of PCSK5. For ( B ), the scale bar is 100 μm. For ( C ), circularities were calculated from N = 1819 ( DCIS.com ) and 2028 (PCSK5 −/− ) segmented spheroids collected from 4 biological replicates at 16 days. Arcsine-transformed circularities were analyzed by two-sample homoscedastic t test. D and E, Multi-cell circularity of PCSK5 −/− cells is restored by wildtype PCSK5 or addition of 250 ng/ml recombinant GDF11, but not PCSK5 M452I or PCSK5 T288P . For ( D ), the scale bar is 100 μm. For ( E ), circularities were calculated from N = 5503 (wildtype PCSK5), 5016 (PCSK5 M452I ), 3495 (PCSK5 T288P ), and 3815 (PCSK5 T288P +GDF11) segmented spheroids collected from 8 biological replicates at 8 days, and arcsine-transformed circularities were analyzed by multiway ANOVA with PCSK5 genotype as a fixed effect. Significant factors were followed up pairwise by Tukey-Kramer post hoc analysis. F and G , PCSK5 alleles do not alter the differentiation phenotypes of MCF10DCIS.com cells in 3D matrigel culture. Cultures in ( A ) plus PCSK5 T288P +GDF11 cultures were lysed and immunoblotted for CDH1, TP63, and VIM with vinculin, tubulin, ERK1/2, and p38 used as loading controls. For ( G ), data from N = 4 biological replicates were normalized to the mean of wildtype PCSK5 cultures, and the three unstimulated genotypes were Box-Cox-transformed and compared by multiway ANOVA with PCSK5 genotype as a fixed effect.

Article Snippet: After 24 hours, conditioned medium was collected, and the concentration of secreted GDF11 was quantified with a GDF11 ELISA (R&D Systems, DY1958).

Techniques: Activity Assay, Transformation Assay, Recombinant