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: Heliyon

Article Title: Astragaloside IV inhibits angiotensin II-induced atrial fibrosis and atrial fibrillation by SIRT1/PGC-1α/FNDC5 pathway

doi: 10.1016/j.heliyon.2024.e30984

Figure Lengend Snippet: AS-IV suppressed the expression of fibrotic genes and SIRT1, PGC-1α and FNDC5 proteins in Ang II-treated mice. (A) The mRNA expression of α-SMA, Collagen I and Collagen III. (B) Representative bands of SIRT1, PGC-1α and FNDC5 by Western blot. (C) Quantification of SIRT1, PGC-1α and FNDC5 proteins. Protein expression was normalized to the expression of GAPDH. (D) Serum irisin concentration in the mice of four groups. Data are presented as mean ± SD (n = 8 in each group). ***p < 0.001 vs. control group; ###p < 0.001 vs. Ang II group. Abbreviation: SIRT1, sirtuin-1; PGC-1α, peroxisome proliferator-activated receptor gamma co-activator 1-alpha; FNDC5, fibronectin type III domain containing 5.

Article Snippet: The following antibodies were incubated on the membrane for one night at 4 °C: FNDC5 (1:400, ab131390, rabbit polyclonal, Abcam), PGC-1α (1:500, ab54481, rabbit polyclonal, Abcam), and GAPDH (1:1000, ab9485, rabbit polyclonal, Abcam).

Techniques: Expressing, Western Blot, Concentration Assay

Journal: Heliyon

Article Title: Astragaloside IV inhibits angiotensin II-induced atrial fibrosis and atrial fibrillation by SIRT1/PGC-1α/FNDC5 pathway

doi: 10.1016/j.heliyon.2024.e30984

Figure Lengend Snippet: Schematic illustration of the protective effects of AS-IV against Ang II-induced atrial fibrosis and AF via upregulation of the SIRT1/PGC-1α/FNDC5 pathway.

Article Snippet: The following antibodies were incubated on the membrane for one night at 4 °C: FNDC5 (1:400, ab131390, rabbit polyclonal, Abcam), PGC-1α (1:500, ab54481, rabbit polyclonal, Abcam), and GAPDH (1:1000, ab9485, rabbit polyclonal, Abcam).

Techniques:

Journal: The European Journal of Orthodontics

Article Title: Irisin reduces orthodontic tooth movement in rats by promoting the osteogenic potential in the periodontal ligament

doi: 10.1093/ejo/cjad021

Figure Lengend Snippet: Confocal images of sagittal sections of maxillary right first molars from control and irisin-treated groups stained with FNDC5, and quantification of immunofluorescence intensity. A. Histological sections of maxillary right first molars were assessed by immunofluorescence staining against FNDC5 (1–3), nuclei were counterstained with DRAQ5 (4–6) and merged images are displayed in 7–9. Boxed areas are shown at a higher magnification, white boxes indicate tension side while red boxes indicate compression side. T: tension side, C: compression side. B. Quantification of FNDC5 fluorescence intensity. Significantly different from control at * P ≤ 0.05 and *** P ≤ 0.001. Mean fluorescence intensities were measured from five random regions of interest for each group, one-way ANOVA was used for statistical analysis.

Article Snippet: The sections were incubated with the following primary antibodies, rabbit anti-vWF (1:500, ab287962, Abcam, Cambridge, UK), mouse anti-collagen type I antibody (1:300, ab90395, Abcam), rabbit anti-periostin antibody (1:300, ab14041, Abcam), mouse anti-OCN antibody (1:200, 33-5400, Thermo Fisher Scientific, Waltham, USA) and rabbit anti-FNDC5 C-terminal antibody (1:200, ab181884, Abcam) in 2 per cent NGS overnight at 4°C.

Techniques: Staining, Immunofluorescence, Fluorescence

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