Journal: Frontiers in Physiology

Article Title: Dysregulated myokines and signaling pathways in skeletal muscle dysfunction in a cigarette smoke–induced model of chronic obstructive pulmonary disease

doi: 10.3389/fphys.2022.929926

Figure Lengend Snippet: Cigarette smoke exposure attenuated fibronectin type III domain-containing protein 5 (Fndc5)/irisin production but augment myostatin (Mstn) expression in skeletal muscles and serum, accompanied by muscle fiber-type switch. (A) boxplot of the statistical results of some myokines and related receptors, such as Mstn, Fndc5, IL-6, TGF-βR1, TGF-βR2 , and TGF-βR3 . (B,C) real-time quantitative PCR examined the expression of Fndc5 and Mstn from gastrocnemius muscle. (D) corplot showing the correlation between Fndc5 and TGF-βR1. (E) WB explored the protein production (left), the ratio of protein to Gapdh (middle), and the ratio of p-Smad3 to Smad3 (right). (F) expression of Fndc5 and Mstn distributed in the gastrocnemius muscle using confocal microscopy. (G,H) levels of irisin and Mstn in serum were detected using ELISA. (I) expression of MyHC (slow) and MyHC (IID) were explored using immunohistochemistry. n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Article Snippet: The primary antibodies, including anti-FNDC5 antibody (bs-8486R, Bioss, Beijing, China) (1:1,000), anti-GDF8/myostatin antibody (ab203076, Abcam, Cambridge, United Kingdom) (1:1,000), antimyosin heavy chain (MyHC)-IID antibody (bs-5885R, Bioss, Beijing, China) (1:1,000), and anti-MYH7 (MyHC (slow)) antibody (GB111857, Servicebio, Wuhan, China) (1:1,000), were incubated with the tissue slides overnight at 4°C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Frontiers in Physiology

Article Title: Dysregulated myokines and signaling pathways in skeletal muscle dysfunction in a cigarette smoke–induced model of chronic obstructive pulmonary disease

doi: 10.3389/fphys.2022.929926

Figure Lengend Snippet: Cigarette smoke extract (CSE) altered myostatin (Mstn) and Fndc5 expression in C2C12 myotubes. (A) cell viability of C2C12 myotubes was tested in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and stimulated with different concentrations of CSE for 24 h. (B) histogram showing the mean fluorescence intensity (MFI) of fibronectin type III domain-containing protein 5 (Fndc5) and myostatin (Mstn) in 3% CSE-stimulated C2C12 myotubes at indicated time points. (C) expression of Fndc5 and Mstn in 3% CSE-stimulated C2C12 myotubes at indicated time points were detected using real-time quantitative PCR (RT-qPCR). (D) WB showing the expression of different proteins in 3% CSE-stimulated C2C12 myotubes. (E) histogram showing the MFI of Fndc5 and Mstn in CSE-stimulated C2C12 myotubes at different concentrations of CSE. (F) expression of Fndc5 and Mstn in CSE-stimulated C2C12 myotubes at different concentrations of CSE were detected using real-time quantitative PCR. (G) WB showing the expression of different proteins in CSE-stimulated C2C12 myotubes at different concentrations of CSE for 24 h. (H) WB showing the expression of different proteins in Mstn and/or ZLN005 (ZLN, 10 μM)-stimulated C2C12 myotubes for 24 h, which also be detected using FACS (I) and RΤ-qPCR (J) . (K) supernatant of cell culture was detected using ELISA. (L) WB showing the expression of different proteins in recombinant irisin (100 ng/ml, up), Mstn (100 ng/ml, down), and/or 3% CSE-stimulated C2C12 myotubes for 24 h, which were also detected using FACS (M) and RΤ-qPCR (N) . n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Article Snippet: The primary antibodies, including anti-FNDC5 antibody (bs-8486R, Bioss, Beijing, China) (1:1,000), anti-GDF8/myostatin antibody (ab203076, Abcam, Cambridge, United Kingdom) (1:1,000), antimyosin heavy chain (MyHC)-IID antibody (bs-5885R, Bioss, Beijing, China) (1:1,000), and anti-MYH7 (MyHC (slow)) antibody (GB111857, Servicebio, Wuhan, China) (1:1,000), were incubated with the tissue slides overnight at 4°C.

Techniques: Expressing, Modification, Fluorescence, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Frontiers in Physiology

Article Title: Dysregulated myokines and signaling pathways in skeletal muscle dysfunction in a cigarette smoke–induced model of chronic obstructive pulmonary disease

doi: 10.3389/fphys.2022.929926

Figure Lengend Snippet: Graphic summary. On the one hand, cigarette smoke extract (CSE) exposure could enhance myostatin (Mstn) production through the Erk1/2 pathway, which further activated the Smad3/PGC-1α pathway, and negatively regulated Fndc5 production. On the other hand, CSE exposure might partially and directly decrease the expression of Fndc5. TEW-7197, a selective TGF-β receptor ALK4/ALK5 inhibitor that can stop Mstn binding to its receptor. SIS3, a specific Smad3 inhibitor by inhibiting Smad3 phosphorylation to suppress Smad3 signaling pathway. U0126, a specific inhibitor of Erk1/2.

Article Snippet: The primary antibodies, including anti-FNDC5 antibody (bs-8486R, Bioss, Beijing, China) (1:1,000), anti-GDF8/myostatin antibody (ab203076, Abcam, Cambridge, United Kingdom) (1:1,000), antimyosin heavy chain (MyHC)-IID antibody (bs-5885R, Bioss, Beijing, China) (1:1,000), and anti-MYH7 (MyHC (slow)) antibody (GB111857, Servicebio, Wuhan, China) (1:1,000), were incubated with the tissue slides overnight at 4°C.

Techniques: Expressing, Binding Assay

Journal: Frontiers in Endocrinology

Article Title: Expression of Recombinant Rat Secretable FNDC5 in Pichia Pastoris and Detection of Its Biological Activity

doi: 10.3389/fendo.2022.852015

Figure Lengend Snippet: r-sFNDC5 induced the browning and lipolysis of 3T3-L1 adipocytes. The biological activity of purified r-sFNDC5 was assessed in differentiated 3T3-L1 adipocytes. (A) 3T3-L1 preadipocytes were treated with various concentrations of r-sFNDC5 for the indicated times, and cell viability was assessed using a CCK-8 assay. The data are expressed as OD values at 450 nm. After fully differentiating, mature 3T3-L1 adipocytes were treated with r-sFNDC5 (20 nM and 50 nM) for 8 h. Then, the relative mRNA levels of browning genes (B) and lipolysis genes (C) were measured by RT–qPCR, and (D, E) western blotting was performed for UCP-1 and HSL. β-actin expression was used as a control. (F) Representative 3T3-L1 adipocytes immunostained for UCP-1 (green) and nuclei (blue) after r-sFNDC5 (50 nM) treatment for 24 h or 4 days. White arrows indicate UCP-1-positive cells. Images were taken using a confocal fluorescence microscope. (G) ATP levels measured in lysates of 3T3-L1 adipocytes treated with 20-100 nM r-sFNDC5 for 4 days. ATP concentrations were normalized to protein content and control. Each experiment was repeated three times. Values are the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control.

Article Snippet: The antibodies used were as follows: FNDC5 (#ab174833, Abcam), UCP-1 (#U6382, Sigma), HSL (#ab109400, Abcam), perilipin (#ab3526, Abcam), adipoq (#ab22554, Abcam), and β-actin (#A5316, Sigma).

Techniques: Activity Assay, Purification, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Expressing, Fluorescence, Microscopy

Journal: Frontiers in Endocrinology

Article Title: Expression of Recombinant Rat Secretable FNDC5 in Pichia Pastoris and Detection of Its Biological Activity

doi: 10.3389/fendo.2022.852015

Figure Lengend Snippet: The function comparison of r-sFNDC5 and r-irisin in the 3T3-L1 adipocytes. After fully differentiated, 3T3-L1 adipocytes were treated with r-sFNDC5 (20 nM) or r-irisin (20 nM) for 8 h. (A) Relative mRNA levels of browning genes, (B) mitochondrial biogenesis, and (C) lipid metabolism were measured by RT–qPCR. (D) The contents of UCP-1 and HSL were measured using western blotting. β-actin expression was used as a control. The asterisk (*) above the bar denotes statistically significant differences in mRNA levels calculated relative to the control, while the hash (#) denotes statistically significant differences calculated between the irisin and sFNDC5 groups. Each experiment was repeated three times. Values are the mean ± SEM. *P < 0.05 vs. control, # P < 0.05 vs. irisin.

Article Snippet: The antibodies used were as follows: FNDC5 (#ab174833, Abcam), UCP-1 (#U6382, Sigma), HSL (#ab109400, Abcam), perilipin (#ab3526, Abcam), adipoq (#ab22554, Abcam), and β-actin (#A5316, Sigma).

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Cell metabolism

Article Title: Detection and Quantitation of Circulating Human Irisin by Tandem Mass Spectrometry

doi: 10.1016/j.cmet.2015.08.001

Figure Lengend Snippet: (A) Schematic representation of the FNDC5 protein structure (top) and irisin (bottom). SP = signal peptide, H = hydrophobic domain, C = c-terminal domain. Human FNDC5 sequence with corresponding domains colored. Human irisin sequence is underlined as well as synthetic AQUA peptides used in this study (red).

Article Snippet: Membranes were then probed with anti-FNDC5 (Irisin) rabbit polyclonal antibody (1:1000) (IN102, Adipogen) overnight at 4 °C, washed with PB ST and incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000) (Sigma) followed by chemiluminescent (Perkin Elmer Life Sciences) detection.

Techniques: Sequencing