Journal: International Journal of Molecular Sciences

Article Title: The Role of Irisin/FNDC5 Expression and Its Serum Level in Breast Cancer

doi: 10.3390/ijms24108628

Figure Lengend Snippet: Comparison of FNDC5/Ir (( A )—IRS score 8) expression with E-cadherin (( B )—IRS score 12), N-cadherin (( C )—IRS score 8), SNAIL (( D )—IRS score 3 and % of nuclear expression 4, ( E )—IRS score 12 and % of nuclear expression 4), SLUG (( F )—IRS score 8 and % of nuclear expression 3) and TWIST (( G )—IRS score 4 and % of nuclear expression 2, ( H )—IRS score 4 and % of nuclear expression 2) using immunohistochemistry (IHC) (positive reactions—brown cell cytoplasm) in breast cancer (BC), magnification ×200.

Article Snippet: The expression of proteins was detected by specific primary antibodies, i.e., polyclonal rabbit: anti-irisin/FNDC5 (dilution 1:50; code no. NBP2-14024; Novus Biologicals, Littleton, CO, USA) and anti-SNAIL (1:400, Clone, code 13099-1-AP, Proteintech, Rosemont, IL, USA), monoclonal mouse: anti-E-cadherin antibody (ready to use, Clone NCH-38, code IR059; Dako, Glostrup, Denmark), anti-N-cadherin (1:50, Clone 6G11, code M3613; Dako), anti-SLUG (1:50, clone A-7, sc-166476, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and monoclonal mouse anti-TWIST (dilution 1:50, clone Twist2C1a, code ab-50887, Abcam, Cambridge, UK).

Techniques: Expressing, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: The Role of Irisin/FNDC5 Expression and Its Serum Level in Breast Cancer

doi: 10.3390/ijms24108628

Figure Lengend Snippet: Correlation of FNDC5/Ir expression level with E-cadherin ( A ) and N-cadherin ( B ), cytoplasmic ( C ) and nuclear ( D ) SNAIL expression levels, cytoplasmic ( E ) and nuclear ( F ) SLUG expression levels, cytoplasmic ( G ) and nuclear ( H ) TWIST expression levels in breast cancer (BC) (sample size n = 541).

Article Snippet: The expression of proteins was detected by specific primary antibodies, i.e., polyclonal rabbit: anti-irisin/FNDC5 (dilution 1:50; code no. NBP2-14024; Novus Biologicals, Littleton, CO, USA) and anti-SNAIL (1:400, Clone, code 13099-1-AP, Proteintech, Rosemont, IL, USA), monoclonal mouse: anti-E-cadherin antibody (ready to use, Clone NCH-38, code IR059; Dako, Glostrup, Denmark), anti-N-cadherin (1:50, Clone 6G11, code M3613; Dako), anti-SLUG (1:50, clone A-7, sc-166476, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and monoclonal mouse anti-TWIST (dilution 1:50, clone Twist2C1a, code ab-50887, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Role of Irisin/FNDC5 Expression and Its Serum Level in Breast Cancer

doi: 10.3390/ijms24108628

Figure Lengend Snippet: Comparison of mRNA FNDC5 expression levels detected by RT-PCR ( A ) and FNDC5/Ir levels ( B ) in the normal breast cell line (Me16c) and different types of BC cell lines (MCF-7, MDA-MB-231, MDA-MB-468) * p < 0.05 ** p < 0.01 *** p < 0.001.

Article Snippet: The expression of proteins was detected by specific primary antibodies, i.e., polyclonal rabbit: anti-irisin/FNDC5 (dilution 1:50; code no. NBP2-14024; Novus Biologicals, Littleton, CO, USA) and anti-SNAIL (1:400, Clone, code 13099-1-AP, Proteintech, Rosemont, IL, USA), monoclonal mouse: anti-E-cadherin antibody (ready to use, Clone NCH-38, code IR059; Dako, Glostrup, Denmark), anti-N-cadherin (1:50, Clone 6G11, code M3613; Dako), anti-SLUG (1:50, clone A-7, sc-166476, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and monoclonal mouse anti-TWIST (dilution 1:50, clone Twist2C1a, code ab-50887, Abcam, Cambridge, UK).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: The Role of Irisin/FNDC5 Expression and Its Serum Level in Breast Cancer

doi: 10.3390/ijms24108628

Figure Lengend Snippet: Comparison of FNDC5/Ir expression by confocal microscopy in the normal breast cell line (Me16c) and different BC cell lines (MCF-7, MDA-MB-231, MDA-MB-468).

Article Snippet: The expression of proteins was detected by specific primary antibodies, i.e., polyclonal rabbit: anti-irisin/FNDC5 (dilution 1:50; code no. NBP2-14024; Novus Biologicals, Littleton, CO, USA) and anti-SNAIL (1:400, Clone, code 13099-1-AP, Proteintech, Rosemont, IL, USA), monoclonal mouse: anti-E-cadherin antibody (ready to use, Clone NCH-38, code IR059; Dako, Glostrup, Denmark), anti-N-cadherin (1:50, Clone 6G11, code M3613; Dako), anti-SLUG (1:50, clone A-7, sc-166476, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and monoclonal mouse anti-TWIST (dilution 1:50, clone Twist2C1a, code ab-50887, Abcam, Cambridge, UK).

Techniques: Expressing, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: The Role of Irisin/FNDC5 Expression and Its Serum Level in Breast Cancer

doi: 10.3390/ijms24108628

Figure Lengend Snippet: Immunolocalization of Ir in transmission electron microscopy. Ultrathin section examination of human adenocarcinoma BC cells (from tumors, ( A – D ) and MDA-MB 468 cell line, ( E , F )). The specific primary antibody against FNDC5/Ir was applied. Next, the ultrathin sections were labeled with the secondary antibody conjugated with the 20 nm-colloidal gold nanoparticles, which shows the antigen distribution in the cells. Arrows indicate positive gold nanoparticles. A strong reaction was detected in the cytoplasm of cancer cells, in the mitochondria, and at the border of the cell membranes of neighboring cells. Note the localization of Ir near the specific microvilli–like structure ( E ) and at the cytoplasmatic processes of BC cells ( F ). Brief double staining with UranyLess solution and lead citrate (3%). IDC—invasive ductal carcinoma at different grades of malignancy, N—nucleus, Mi—mitochondrion.

Article Snippet: The expression of proteins was detected by specific primary antibodies, i.e., polyclonal rabbit: anti-irisin/FNDC5 (dilution 1:50; code no. NBP2-14024; Novus Biologicals, Littleton, CO, USA) and anti-SNAIL (1:400, Clone, code 13099-1-AP, Proteintech, Rosemont, IL, USA), monoclonal mouse: anti-E-cadherin antibody (ready to use, Clone NCH-38, code IR059; Dako, Glostrup, Denmark), anti-N-cadherin (1:50, Clone 6G11, code M3613; Dako), anti-SLUG (1:50, clone A-7, sc-166476, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and monoclonal mouse anti-TWIST (dilution 1:50, clone Twist2C1a, code ab-50887, Abcam, Cambridge, UK).

Techniques: Transmission Assay, Electron Microscopy, Labeling, Double Staining

Journal: International Journal of Molecular Sciences

Article Title: The Role of Irisin/FNDC5 Expression and Its Serum Level in Breast Cancer

doi: 10.3390/ijms24108628

Figure Lengend Snippet: Immunolocalization of Ir in transmission electron microscopy. Ultrathin section examination of human adenocarcinoma BC cells from the breast tumor microenvironment (stroma). All electronograms show invasive ductal carcinoma G2. The specific primary antibody against FNDC5/Ir was applied as previously described, followed by applying the species-specific secondary antibody conjugated with 20 nm-colloidal gold nanoparticles. Arrows indicate positive gold nanoparticles. Strong immunogold reaction was detected in the extracellular matrix and cancer-associated fibroblasts (in the cytoplasm and at the border of the cell membrane). Brief double staining with UranyLess solution and lead citrate (3%). N—nucleus, Gs—ground substance of the extracellular matrix ( A ), Cf—collagen fibers, RER—rough endoplasmic reticulum in the fibroblast ( B ). IF—intermediate filaments ( C ), Mi—mitochondrion ( D ).

Article Snippet: The expression of proteins was detected by specific primary antibodies, i.e., polyclonal rabbit: anti-irisin/FNDC5 (dilution 1:50; code no. NBP2-14024; Novus Biologicals, Littleton, CO, USA) and anti-SNAIL (1:400, Clone, code 13099-1-AP, Proteintech, Rosemont, IL, USA), monoclonal mouse: anti-E-cadherin antibody (ready to use, Clone NCH-38, code IR059; Dako, Glostrup, Denmark), anti-N-cadherin (1:50, Clone 6G11, code M3613; Dako), anti-SLUG (1:50, clone A-7, sc-166476, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and monoclonal mouse anti-TWIST (dilution 1:50, clone Twist2C1a, code ab-50887, Abcam, Cambridge, UK).

Techniques: Transmission Assay, Electron Microscopy, Double Staining

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

Journal: Scientific Reports

Article Title: Irisin – a myth rather than an exercise-inducible myokine

doi: 10.1038/srep08889

Figure Lengend Snippet: Polyclonal irisin antibodies used in this and related ELISA kits

Article Snippet: The following antibodies were used in this study: pAb-A, rabbit polyclonal antibody (pAb) to irisin (Phoenix Europe, Karlsruhe, Germany); pAb-B, rabbit pAb to irisin (Cayman Chemicals, Ann Arbor, USA); pAb-C, rabbit pAb to irisin (AdipoGen, Liestal, Switzerland); pAb-D, rabbit pAb to irisin (Phoenix Europe); and pAb-E, rabbit pAb to FNDC5 (C-terminal; BioCat, Heidelberg, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Affinity Purification

Journal: International Journal of Molecular Sciences

Article Title: Cerebral Benefits Induced by Electrical Muscle Stimulation: Evidence from a Human and Rat Study

doi: 10.3390/ijms25031883

Figure Lengend Snippet: Effect of EMS on the FNDC5/irisin pathway. ( A ) Relative protein levels of FNDC5/irisin in quadriceps muscle at 4 h (hatched bar) and 24 h (full bar) in the SHAM (black) and EMS (green) groups. ( B ) Spearman correlation between hippocampal BDNF and quadriceps muscle FNDC5/irisin relative protein expression levels at the 24 h time point for SHAM rats (black circle) and EMS rats (green triangle). ( C , D ) Circulating irisin levels measured by ELISA immediately (tail blood collection, ( C )) and 4 or 24 h (intracardiac blood collection, ( D )) after protocol application. ( E ) Representative immunoblots of pooled immunoprecipitated serum immediately, 4 h, and 24 h following the procedures. ( F ) Schematic signaling pathway activated by irisin upon binding to the integrin receptor. ( G ) Relative protein levels of FNDC5/irisin in the hippocampus 4 and 24 h after the procedures. ( H ) Relative protein levels of p-FAK Tyr397 in the hippocampus at the 4 h and 24 h time points. Corresponding immunoblots are shown on the side of the graphs. NS means no significance.

Article Snippet: FNDC5/irisin , Abcam recombinant anti-FNDC5 antibody rabbit monocolonal [EPR12209] (ab174833) , 1/3000 TBST-Milk (5%).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunoprecipitation, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Cerebral Benefits Induced by Electrical Muscle Stimulation: Evidence from a Human and Rat Study

doi: 10.3390/ijms25031883

Figure Lengend Snippet: Primary antibodies.

Article Snippet: FNDC5/irisin , Abcam recombinant anti-FNDC5 antibody rabbit monocolonal [EPR12209] (ab174833) , 1/3000 TBST-Milk (5%).

Techniques: Recombinant, Transduction, Purification

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