Journal: Journal of cellular physiology

Article Title: Glial-derived neurotrophic factor in human airway smooth muscle.

doi: 10.1002/jcp.30489

Figure Lengend Snippet: FIGURE 1 Glial‐derived neurotrophic factor family of ligands (GFLs) and their receptors in airways. Glial‐derived neurotrophic factor (GDNF) is expressed in whole lung, with greater expression in lungs of mild‐moderate asthmatics (a). In non‐asthmatic airway smooth muscle (ASM) cells, the GFLs ‐GDNF (b) and neurturin (NRTN; c) are expressed, with pro‐inflammatory cytokines such as TNF‐α or IL‐13 increasing them, albeit to different extents. ASM cells also express the GFL receptors Ret (d), GFRα1 (e) and GFRα2 (f), with cytokines increasing only GFRα1. *p < .05, versus normal or control. Data represented as mean ± SEM; N = 9–11 for (b)–(f), and N = 19 for non‐asthmatics and N = 5 asthmatics in (a). *indicates significant effect (p < .05). IL, interleukin; SEM, standard error of mean; TNF‐α, tumor necrosis factor‐α

Article Snippet: Pharmacological modulators included recombinant human GDNF (rhGDNF) and NRTN (rhNRTN) (Cat# 212‐GD and 1297‐NE respectively, R&D systems), and Ret inhibitor SPP86 (Cat# SML1435) was from Tocris.

Techniques: Derivative Assay, Expressing, Control

Journal: Journal of cellular physiology

Article Title: Glial-derived neurotrophic factor in human airway smooth muscle.

doi: 10.1002/jcp.30489

Figure Lengend Snippet: FIGURE 2 Acute effect of GDNF and NRTN on [Ca2+]i responses in human ASM: Nonasthmatic ASM cells were treated with GDNF (a) or NRTN (b) in concentrations ranging from 1 ng/ml to 100 ng/ml. Acute treatment (15 min) with GDNF or NRTN induced varying degrees of transient [Ca2+]i responses. Amplitude of [Ca2+]i response was increased by mid‐concentrations of either ligand. Data represented as mean ± SEM from N of 4 patients each. *indicates significant effect (p < .05). ASM, airway smooth muscle; GDNF, glial‐derived neurotrophic factor; NRTN, neurturin; SEM, standard error of mean

Article Snippet: Pharmacological modulators included recombinant human GDNF (rhGDNF) and NRTN (rhNRTN) (Cat# 212‐GD and 1297‐NE respectively, R&D systems), and Ret inhibitor SPP86 (Cat# SML1435) was from Tocris.

Techniques: Derivative Assay

Journal: Journal of cellular physiology

Article Title: Glial-derived neurotrophic factor in human airway smooth muscle.

doi: 10.1002/jcp.30489

Figure Lengend Snippet: FIGURE 3 Enhancing effect of GDNF on agonist‐induced [Ca2+]i responses in ASM cells: In nonasthmatic human ASM cells, short‐term pretreatment with GDNF (15 min) enhanced subsequent [Ca2+]i responses to the bronchoconstrictor agonists ACh (a) or histamine (b) compared with the agonist alone. In contrast, pretreatment with NRTN did not have an enhancing effect (c) and (d). Data represented as mean ± SEM from N of 4–5 patients each. *indicates significant effect (p < .05). ACh, acetylcholine; ASM, airway smooth muscle; GDNF, glial‐derived neurotrophic factor; NRTN, neurturin; SEM, standard error of mean

Article Snippet: Pharmacological modulators included recombinant human GDNF (rhGDNF) and NRTN (rhNRTN) (Cat# 212‐GD and 1297‐NE respectively, R&D systems), and Ret inhibitor SPP86 (Cat# SML1435) was from Tocris.

Techniques: Derivative Assay

Journal: Journal of cellular physiology

Article Title: Glial-derived neurotrophic factor in human airway smooth muscle.

doi: 10.1002/jcp.30489

Figure Lengend Snippet: FIGURE 4 Role of Ret and GFRαs in GDNF and NRTN effects on [Ca2+]i in ASM: Nonasthmatic human ASM cells were subjected to GDNF (a) or NRTN (b) treatment in the presence or absence of Ret inhibitor (SPP86;10 µM; 24 h) or GFR chelators (GFRα1‐Fc vs. GFRα2‐Fc; 1 µg/ml; 24 h) and [Ca2+]i response were measured. Ret inhibition and GFRα1‐Fc both significantly blunted GDNF effects while Ret inhibition or GFRα2‐ Fc were less effective in blunting NRTN effects, albeit significant. Data represented as mean ± SEM from N of 3–4 patients each. *indicates significant effect (p < .05). ASM, airway smooth muscle; GDNF, glial‐derived neurotrophic factor; NRTN, neurturin; SEM, standard error of mean

Article Snippet: Pharmacological modulators included recombinant human GDNF (rhGDNF) and NRTN (rhNRTN) (Cat# 212‐GD and 1297‐NE respectively, R&D systems), and Ret inhibitor SPP86 (Cat# SML1435) was from Tocris.

Techniques: Inhibition, Derivative Assay

Journal: Journal of cellular physiology

Article Title: Glial-derived neurotrophic factor in human airway smooth muscle.

doi: 10.1002/jcp.30489

Figure Lengend Snippet: FIGURE 6 Effect of ASM‐derived GDNF on [Ca2+]i response to histamine: GDNF in nonasthmatic and asthmatic human ASM conditioned media were quantified by ELISA (a). Amplitude of histamine‐induced [Ca2+]i response was reduced with 24 h pre‐exposure to Ret inhibitor SPP86 (b) or GFR α1‐Fc (c) highlighting an autocrine effect of secreted GDNF on ASM [Ca2+]i. Data represented as mean ± SEM from N of 5–7 patients each. *indicates significant effect (p < .05). ASM, airway smooth muscle; ELISA, enzyme‐linked immunosorbent assay; GDNF, glial‐derived neurotrophic factor; NRTN, neurturin; SEM, standard error of mean

Article Snippet: Pharmacological modulators included recombinant human GDNF (rhGDNF) and NRTN (rhNRTN) (Cat# 212‐GD and 1297‐NE respectively, R&D systems), and Ret inhibitor SPP86 (Cat# SML1435) was from Tocris.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of cellular physiology

Article Title: Glial-derived neurotrophic factor in human airway smooth muscle.

doi: 10.1002/jcp.30489

Figure Lengend Snippet: FIGURE 5 Chronic effect of GDNF and NRTN on [Ca2+]i response to histamine: (a) Representative traces showing [Ca2+]i response to histamine in nonasthmatic ASM cells following 24 h exposure to GDNF, NRTN, or vehicle. (b) The amplitude of [Ca2+]i responses to histamine were increased by chronic pre‐exposure to GDNF but not NRTN. Data represented as mean ± SEM from N of 5 patients each. *indicates significant effect (p < .05). GDNF, glial‐derived neurotrophic factor; NRTN, neurturin; SEM, standard error of mean

Article Snippet: Pharmacological modulators included recombinant human GDNF (rhGDNF) and NRTN (rhNRTN) (Cat# 212‐GD and 1297‐NE respectively, R&D systems), and Ret inhibitor SPP86 (Cat# SML1435) was from Tocris.

Techniques: Derivative Assay

Journal: Journal of cellular physiology

Article Title: Glial-derived neurotrophic factor in human airway smooth muscle.

doi: 10.1002/jcp.30489

Figure Lengend Snippet: FIGURE 8 Schematic of GDNF and NRTN effects in ASM. Human ASM can produce GDNF family ligands (GFLs) such as GDNF and NRTN that can have autocrine/paracrine effects by inducing [Ca2+]i responses by themselves, and promoting responses to agonist‐ induced [Ca2+]i, thus promoting contractility. ASM, airway smooth muscle; GDNF, glial‐derived neurotrophic factor; NRTN, neurturin

Article Snippet: Pharmacological modulators included recombinant human GDNF (rhGDNF) and NRTN (rhNRTN) (Cat# 212‐GD and 1297‐NE respectively, R&D systems), and Ret inhibitor SPP86 (Cat# SML1435) was from Tocris.

Techniques: Derivative Assay

Journal: Journal of cellular physiology

Article Title: Glial-derived neurotrophic factor in human airway smooth muscle.

doi: 10.1002/jcp.30489

Figure Lengend Snippet: FIGURE 7 Effect of GFLs on histamine‐induced myosin light chain (MLC) phosphorylation: Human ASM cells were pretreated with GDNF or NRTN for 24 h, followed by stimulation with 1 µM histamine (Hist) for 5 min. Cells were lysed and immunoblotted for pMLC and β‐actin for loading control. Chronic GDNF treatment increased the histamine‐induced phosphorylation of MLC compared with vehicle. Data represented as mean ± SEM from N of six patients each. *p < .05, ***p < .001 versus vehicle (−Histamine). #p < .05 versus vehicle (+Histamine). ASM, airway smooth muscle; GDNF, glial‐ derived neurotrophic factor; GFLs, glial‐derived neurotrophic factor family of ligands; NRTN, neurturin; SEM, standard error of mean

Article Snippet: Pharmacological modulators included recombinant human GDNF (rhGDNF) and NRTN (rhNRTN) (Cat# 212‐GD and 1297‐NE respectively, R&D systems), and Ret inhibitor SPP86 (Cat# SML1435) was from Tocris.

Techniques: Phospho-proteomics, Control, Derivative Assay

Journal: eLife

Article Title: NHE6 depletion corrects ApoE4-mediated synaptic impairments and reduces amyloid plaque load

doi: 10.7554/eLife.72034

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , γ-Secretase inhibitor L-685458 , Tocris Bioscience , 2627 , .

Techniques: Staining, Plasmid Preparation, Transfection, Construct, Expressing, shRNA, Recombinant, Sequencing, Software, Imaging

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