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: Bone & Joint Research

Article Title: GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis

doi: 10.1302/2046-3758.117.BJR-2022-0019.R1

Figure Lengend Snippet: Gene primer sequences for mouse used for quantitative real-time polymerase chain reaction.

Article Snippet: GDF11 (10 μM, P6319, Abnova, USA) was injected to UAC with GDF11 injection groups and the same volume of vehicle to UAC with vehicle injection group.

Techniques:

Journal: Bone & Joint Research

Article Title: GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis

doi: 10.1302/2046-3758.117.BJR-2022-0019.R1

Figure Lengend Snippet: Primer sequences for rat used for quantitative real-time polymerase chain reaction.

Article Snippet: GDF11 (10 μM, P6319, Abnova, USA) was injected to UAC with GDF11 injection groups and the same volume of vehicle to UAC with vehicle injection group.

Techniques:

Journal: Bone & Joint Research

Article Title: GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis

doi: 10.1302/2046-3758.117.BJR-2022-0019.R1

Figure Lengend Snippet: Abnormal adipogenesis in condylar cartilage of mice with temporomandibular joint (TMJ) osteoarthritis (OA) induced by unilateral anterior crossbite (UAC) stimulation. a) Immunohistochemical staining (IHC) of Adiponectin (AdipoQ) in the TMJs of sham and UAC mice. b) The proportion of AdipoQ-positive chondrocytes and messenger RNA (mRNA) expression of AdipoQ were increased in the TMJs of UAC mice. c) IHC of peroxisome proliferator-activated receptor γ (PPARγ) in the TMJs of sham and UAC mice. d) The proportion of PPARγ-positive chondrocytes and mRNA expression of PPARγ were increased in the TMJs of UAC mice. e) IHC of growth differentiation factor 11 (GDF11) in the TMJs of sham and UAC mice. f) The proportion of GDF11-positive chondrocytes and mRNA expression of GDF11 were decreased in the TMJs of UAC mice. Sham, sham group; 3 WK, 3 weeks; 7 WK, 7 weeks; 11 WK, 11 weeks. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the age-matched sham group, independent-samples t -test .

Article Snippet: GDF11 (10 μM, P6319, Abnova, USA) was injected to UAC with GDF11 injection groups and the same volume of vehicle to UAC with vehicle injection group.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Bone & Joint Research

Article Title: GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis

doi: 10.1302/2046-3758.117.BJR-2022-0019.R1

Figure Lengend Snippet: Fluid flow shear stress (FFSS) promoted abnormal adipogenesis in cultured condylar chondrocytes and decreased the expression of growth differentiation factor 11 (GDF11). a) FFSS decreased the messenger RNA (mRNA) expression of type II collagen ( Col-II ) and aggrecan and increased expression of type X collagen ( Col-X ), alkaline phosphatase ( Alp ), matrix metallopeptidase 13 ( Mmp13 ), peroxisome proliferator-activated receptor γ ( PPARγ ), CCAAT/enhancer-binding protein α ( Cebpα ), fatty acid binding protein 4 ( Fabp4 ), Perilipin1 , and Adiponectin ( Adipoq ) under adipogenic induction medium for 21 days. b) FFSS promoted the formation of lipid droplets in cultured condylar chondrocytes under adipogenic induction medium for 21 days. c) FFSS promoted the protein levels of AdipoQ and PPARγ in the cytoplasm of cultured condylar chondrocytes when cultured in adipogenic induction medium for 21 days. d) and e) FFSS decreased the d) protein level and e) mRNA expression of GDF11 in the cytoplasm of cultured condylar chondrocytes when cultured in adipogenic induction medium for 21 days. f) FFSS induced the increase of PPARγ protein and the decrease of its post-translational modification by small ubiquitin-related modifier (SUMOylation) in cultured condylar chondrocytes when cultured in adipogenic induction medium for 21 days. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the 0 dyne/cm 2 group, one-way analysis of variance (ANOVA). DAPI, 4′,6-diamidino-2-phenylindole; SUMO, small ubiquitin-related modifier.

Article Snippet: GDF11 (10 μM, P6319, Abnova, USA) was injected to UAC with GDF11 injection groups and the same volume of vehicle to UAC with vehicle injection group.

Techniques: Shear, Cell Culture, Expressing, Binding Assay, Modification, Ubiquitin Proteomics

Journal: Bone & Joint Research

Article Title: GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis

doi: 10.1302/2046-3758.117.BJR-2022-0019.R1

Figure Lengend Snippet: Exogenous growth differentiation factor 11 (GDF11) alleviated the abnormal adipogenesis and differentiation of condylar chondrocytes induced by fluid flow shear stress (FFSS). a) Exogenous GDF11 increased the messenger RNA (mRNA) expression of type II collagen ( Col-II ) and Aggrecan , and decreased the mRNA expression of type X collagen ( Col-X ), alkaline phosphatase ( ALP ), matrix metallopeptidase 13 ( Mmp13 ), CCAAT/enhancer-binding protein α (Cebpα), fatty acid binding protein 4 ( Fabp4 ), Perilipin1 , and Adiponectin ( Adipoq ) after FFSS stimulation in condylar chondrocytes when cultured in adipogenic induction medium for 21 days. However, it did not affect the changes in peroxisome proliferator-activated receptor γ ( PPARγ ) expression induced by FFSS. b) Exogenous GDF11 decreased the protein level of AdipoQ but had no influence on PPARγ expression in the cytoplasm of cultured condylar chondrocytes with adipogenic induction medium after FFSS stimulation. c) Exogenous GDF11 inhibited the enhanced formation of lipid droplets induced by FFSS in cultured condylar chondrocytes under adipogenic induction medium for 21 days. d) Exogenous GDF11 did not affect the FFSS-induced increase in PPARγ levels in the cytoplasm of cultured condylar chondrocytes, but it did significantly promote the post-translational modification by small ubiquitin-related modifier (SUMOylation) of PPARγ in chondrocytes when cultured in adipogenic induction medium for 21 days. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the FFSS + vehicle group, one-way analysis of variance (ANOVA). DAPI, 4′,6-diamidino-2-phenylindole; SUMO, small ubiquitin-related modifier.

Article Snippet: GDF11 (10 μM, P6319, Abnova, USA) was injected to UAC with GDF11 injection groups and the same volume of vehicle to UAC with vehicle injection group.

Techniques: Shear, Expressing, Binding Assay, Cell Culture, Modification, Ubiquitin Proteomics

Journal: Bone & Joint Research

Article Title: GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis

doi: 10.1302/2046-3758.117.BJR-2022-0019.R1

Figure Lengend Snippet: Effect of local injection of growth differentiation factor 11 (GDF11) on cartilage degeneration and abnormal adipogenesis of condylar cartilage in the temporomandibular joints (TMJs) of mice. a) Cartilage degeneration of the TMJ was alleviated in unilateral anterior crossbite (UAC) mice injected with GDF11. b) The thickness of the condylar cartilage and safranine O positive area were significantly increased in the TMJs of UAC mice injected with GDF11. c) Immunohistochemical staining of Adiponectin (AdipoQ) and peroxisome proliferator-activated receptor γ (PPARγ) in the TMJs of UAC + vehicle-injected and UAC + GDF11-injected mice. d) The proportion of AdipoQ-positive chondrocytes and messenger RNA (mRNA) expression of Adipo q were decreased in the TMJs of UAC mice injected with GDF11. However, GDF11 injection did not affect the change in PPARγ in the TMJs of mice under UAC stimulation. e) GDF11 injection increased the mRNA expression of type II collagen ( Col-II ) and Aggrecan , and decreased the mRNA expression of type X collagen ( Col-X ), alkaline phosphatase (Alp), matrix metallopeptidase 13 ( Mmp13 ), Adipoq , CCAAT/enhancer-binding protein α ( Cebpα ), fatty acid binding protein 4 ( Fabp4 ), and Perilipin1 in the TMJs of UAC mice. Vehicle, vehicle injection group; GDF11, GDF11 injection group; 7 WK, 7 weeks; 11 WK, 11 weeks. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the age-matched UAC + vehicle injection group, independent-samples t -test .

Article Snippet: GDF11 (10 μM, P6319, Abnova, USA) was injected to UAC with GDF11 injection groups and the same volume of vehicle to UAC with vehicle injection group.

Techniques: Injection, Immunohistochemical staining, Staining, Expressing, Binding Assay

Journal: OncoTargets and therapy

Article Title: GDF11 restrains tumor growth by promoting apoptosis in pancreatic cancer

doi: 10.2147/OTT.S181792

Figure Lengend Snippet: The mRNA and protein expression of GDF11 in pancreatic cancer cell lines and clinical samples. Notes: ( A ) qRT-PCR analysis of GDF11 levels in pancreatic cancer tissues and normal pancreatic tissues (n=28). ( B ) Protein levels of GDF11 were detected by Western blot in eight pairs of pancreatic cancer tissues and normal pancreatic tissues. ( C ) The mRNA expressions of GDF11 in the normal pancreatic cell line HPDE6-C7 and five pancreatic cancer cell lines were examined by qRT-PCR. ( D ) Western blot analysis of GDF11 in the HPDE6-C7 and five pancreatic cancer cell lines. ( E ) Representative images and its regional magnification of GDF11 IHC in pancreatic cancer tissues and their paired normal tissues. a: Low expression of GDF11 in pancreatic cancer tissues; b: high expression of GDF11 in the cytoplasm of normal pancreatic tissues. Data shown represent the mean ± SD. ** P <0.01. *** P <0.001. Abbreviations: GDF11, growth differentiation factor 11; IHC, immunohistochemistry; PC, pancreatic cancer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: These results are in accordance with prognostic summary of GDF11 in pancreatic cancer in the Human Protein Atlas database.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: OncoTargets and therapy

Article Title: GDF11 restrains tumor growth by promoting apoptosis in pancreatic cancer

doi: 10.2147/OTT.S181792

Figure Lengend Snippet: The relationship between GDF11 expression and clinicopathological characteristics in tissue microarray of 63 pancreatic cancer patients

Article Snippet: These results are in accordance with prognostic summary of GDF11 in pancreatic cancer in the Human Protein Atlas database.

Techniques: Expressing, Microarray

Journal: OncoTargets and therapy

Article Title: GDF11 restrains tumor growth by promoting apoptosis in pancreatic cancer

doi: 10.2147/OTT.S181792

Figure Lengend Snippet: Relationship between GDF11 expression and OS in tissue microarray of pancreatic cancer. Notes: ( A ) Representative IHC staining images of GDF11 of paired tumor and nontumor tissues. ( B ) Kaplan–Meier analysis showed that patients with low expression of GDF11 had a poorer OS than those with high expression of GDF11. P =0.012, log-rank test. Abbreviations: GDF11, growth differentiation factor 11; IHC, immunohistochemistry; OS, overall survival; PC, pancreatic cancer.

Article Snippet: These results are in accordance with prognostic summary of GDF11 in pancreatic cancer in the Human Protein Atlas database.

Techniques: Expressing, Microarray, Immunohistochemistry

Journal: OncoTargets and therapy

Article Title: GDF11 restrains tumor growth by promoting apoptosis in pancreatic cancer

doi: 10.2147/OTT.S181792

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of factors associated with overall survival

Article Snippet: These results are in accordance with prognostic summary of GDF11 in pancreatic cancer in the Human Protein Atlas database.

Techniques:

Journal: OncoTargets and therapy

Article Title: GDF11 restrains tumor growth by promoting apoptosis in pancreatic cancer

doi: 10.2147/OTT.S181792

Figure Lengend Snippet: Overexpression of GDF11 suppresses tumor growth and metastasis while knockdown of GDF11 promoted these functions in pancreatic cancer cell line. Notes: PANC-1 cell was transfected using a lentiviral transduction system to enhance GDF11 expression, and CFPAC-1 cell was treated using siRNA to inhibit GDF11 expression. ( A ) Elevated and downregulated GDF11 expression was examined by Western blot and qRT-PCR analyses. ( B ) MTS assay revealed the effects of GDF11 overexpression and knockdown on the proliferation of pancreatic cancer cell lines. ( C, D ) Representative photographs and cell quantifications of transwell migration and invasion assays of elevated and downregulated GDF11 in pancreatic cancer cell lines. Data shown represent the mean ± SD. *** P <0.001. Abbreviations: GDF11, growth differentiation factor 11; LV, lentiviral vectors; NC, negative control; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.

Article Snippet: These results are in accordance with prognostic summary of GDF11 in pancreatic cancer in the Human Protein Atlas database.

Techniques: Over Expression, Knockdown, Transfection, Transduction, Expressing, Western Blot, Quantitative RT-PCR, MTS Assay, Migration, Negative Control

Journal: OncoTargets and therapy

Article Title: GDF11 restrains tumor growth by promoting apoptosis in pancreatic cancer

doi: 10.2147/OTT.S181792

Figure Lengend Snippet: Enhanced GDF11 expression promoted apoptosis and downregulated GDF11 expression inhibited apoptosis in pancreatic cancer cell lines. Notes: ( A ) The effect of GDF11 overexpression on the apoptosis of pancreatic cancer cell lines by flow cytometry. ( B ) The effect of knockdown GDF11 on the apoptosis of pancreatic cancer cell line by flow cytometry. Data shown represent the mean ± SD. *** P <0.001. Abbreviations: GDF11, growth differentiation factor 11; LV, lentiviral vectors; NC, negative control.

Article Snippet: These results are in accordance with prognostic summary of GDF11 in pancreatic cancer in the Human Protein Atlas database.

Techniques: Expressing, Over Expression, Flow Cytometry, Knockdown, Negative Control