Journal: Obesity Facts

Article Title: Genetic Variants in the Activation of the Brown-Like Adipocyte Pathway and the Risk for Severe Obesity

doi: 10.1159/000505666

Figure Lengend Snippet: A Electropherograms of the FNDC5 gene. BFNDC5 structure and position of the 5 mutations identified by Sanger sequencing. C A portion of the amino acid sequence of FNDC5 in diverse species. The position of the new mutation is showed in the red squares, suggesting it is highly conserved in different organisms. The nonconserved position in specific species is shown in the white squares.

Article Snippet: Bioinformatic Tools FNDC5 sequences (genomic, transcript, and protein) were obtained from the National Centre for Biotechnology Information ( https://www.ncbi.nlm.nih.gov/ ) and the Ensembl database ( http://www.ensembl.org/ ).

Techniques: Sequencing, Mutagenesis

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: Nature

Article Title: A PGC1α-dependent myokine that drives browning of white fat and thermogenesis

doi: 10.1038/nature10777

Figure Lengend Snippet: a , qPCR against indicated genes in skeletal muscle from MCK-PGC1α transgenic mice or littermate controls (n=7 from each group). b , qPCR against indicated genes in skeletal muscle from sedentary mice or mice given three weeks of free wheel running (n=10 from each group). Mice were rested for 12 hours prior to euthanization. c , mRNA expression levels from human muscle biopsies before and after 10 weeks of endurance exercise training (8 subjects included). All data points are normalized to baseline levels. d , SVF from the inguinal fat depot, differentiated into adipocytes for 6 days in the presence of saline or recombinant Fndc5 (20 nM), IL-15 (10μM) or VEGFβ (50μM). The graph show normalized mRNA levels of indicated genes. This experiment was repeated three times with similar results. For figure 2d, we performed one-way ANOVA tests where p<0.05 for the effect of Fndc5 on UCP-1 and Cidea expression. All other statistics were performed using students T-test and bar graphs are mean +/− SEM.

Article Snippet: Recombinant Fndc5, Lrg1, IL-15, VEGFβ and TIMP4 were from ABNOVA (Taiwan).

Techniques: Transgenic Assay, Expressing, Saline, Recombinant

Journal: Nature

Article Title: A PGC1α-dependent myokine that drives browning of white fat and thermogenesis

doi: 10.1038/nature10777

Figure Lengend Snippet: a , SVF from the inguinal fat depot was differentiated into adipocytes for 6 days in the presence of saline, recombinant Fndc5 (20 nM), or BMP-7 (3.3 μM). The graph show normalized mRNA levels for indicated genes. Similar results were obtained in more than 10 experiments with the fold induction of UCP1 between 7–500 fold. b , mRNA levels of UCP1 from inguinal-derived SVF treated with Fndc5 for 6 days at indicated doses. c , Clark electrode measurements of oxygen consumption in SVF from the inguinal fat depot, differentiated into adipocytes for 6 days in the presence of saline or recombinant Fndc5 (20 nM). Data is representative for three independent experiments and normalized to total cellular protein. d , qPCR of UCP1 mRNA from SVF, differentiated into adipocytes, and treated with Fndc5 or saline for 6 days followed by addition of forskolin for 8 hours. § indicates p<0.05 compared to forskolin treatment. e , qPCR of PPARα after 6 days of Fndc5 treatment (20 nM) during differentiation of primary SVF. f , SVF, differentiated into adipocytes, and treated with Fndc5 and/or GW6471 for 6 days. The graph shows qPCR of indicated genes, and § indicates p<0.05 compared to Fndc5 treatment. For 3d and f; combined one and two-way ANOVA was used. All other statistics were performed using students T-test and bar graphs are mean +/− SEM.

Article Snippet: Recombinant Fndc5, Lrg1, IL-15, VEGFβ and TIMP4 were from ABNOVA (Taiwan).

Techniques: Saline, Recombinant, Derivative Assay

Journal: Nature

Article Title: A PGC1α-dependent myokine that drives browning of white fat and thermogenesis

doi: 10.1038/nature10777

Figure Lengend Snippet: a , Schematic representation of the Fndc5 protein structure (top), flag-tagged Fndc5 protein (middle) and irisin (bottom). SP = signal peptide, H = hydrophobic domain, C = c-terminal domain, Flag = FLAG epiotope. b , 293 cells transfected with a vector expressing the c-terminal flag tagged Fndc5 (CTF-F5, third panel from a ), followed by isolation of cell and culture media protein. Samples were adjusted for protein content and western blots were performed against the FLAG antigen (left) or Fndc5 (right). This was repeated in several experiments with similar results. Adjusting for volume (instead of protein content) also gave similar results. C = cell fraction and M = media fraction. c , 293 cells transfected with a vector expressing Fndc5-CTF, followed by isolation of cell and media protein. Respective protein fraction was then treated with PNGase F followed by western blot against Fndc5 after SDS-PAGE. d , representation of the full-length Fndc5 and the irisin fragment mapped by mass spectrometry (bold and underlined).

Article Snippet: Recombinant Fndc5, Lrg1, IL-15, VEGFβ and TIMP4 were from ABNOVA (Taiwan).

Techniques: Transfection, Plasmid Preparation, Expressing, Isolation, Western Blot, SDS Page, Mass Spectrometry

Journal: Nature

Article Title: A PGC1α-dependent myokine that drives browning of white fat and thermogenesis

doi: 10.1038/nature10777

Figure Lengend Snippet: a , Plasma from mice injected intravenously with adenoviral vectors expressing Fndc5 or GFP was subjected to western blot against Fndc5. b , western blot against irisin in plasma from muscle-specific PGC1α knockout (MKO) mice or control littermates (flox/flox). c , western blot against irisin in plasma from wild-type mice or two healthy human subjects (representative for 8 subjects analyzed identically). d , western blot against irisin in serum from control or three weeks exercised mice, followed by 12h rest. Bottom panel shows quantification of the bands. e , western blot analysis of irisin in plasma from human subjects before and after 10 weeks of endurance exercise. 8 subjects in total were analyzed; quantification after internal normalization is displayed in bottom panel. For all plasma analyses, samples were depleted for albumin/IgG, and deglycosylated using PNGase F. Arrow indicated irisin band. Data is presented as mean +/−SEM, and * p<0.05 compared to control group. Students T-test was used for single comparisons.

Article Snippet: Recombinant Fndc5, Lrg1, IL-15, VEGFβ and TIMP4 were from ABNOVA (Taiwan).

Techniques: Clinical Proteomics, Injection, Expressing, Western Blot, Knock-Out, Control

Journal: Nature

Article Title: A PGC1α-dependent myokine that drives browning of white fat and thermogenesis

doi: 10.1038/nature10777

Figure Lengend Snippet: a–c , Wild-type BALB/c mice were injected with 10 10 GFP or Fndc5-expressing adenoviral particles intravenously (n=7 for each group). Animals were sacrificed after 10 days and inguinal/subcutaneous fat pads were collected and analyzed using qPCR analysis of indicated mRNAs ( a ) and western blot against UCP1 ( b ). c , representative images from immunohistochemistry against UCP1 in these mice. All results in a–c were repeated 2 times with similar results. d–g , C57/Bl6 mice fed a 60% kcal high fat diet for 20 weeks were intravenously injected with GFP or Fndc5-expressing adenovirus and all analyses were done 10 days thereafter (n=7 for both groups). d . oxygen consumption at day and night. e , body weights of 10 days after injection with indicated adenovirus in these mice. f , fasting plasma insulin measured using ELISA. g , intraperitoneal glucose tolerance test. h , mice were injected IP with 50μg of rabbit IgG or a rabbit anti-Fndc5 antibody and were either subjected to swimming for 7 days or kept sedentary (n=10 for all groups). Data displays mRNA expression levels from inguinal WAT. All data in d–j were performed at least twice in a separate mouse cohort with similar results. § p<0.05 compared to exercise + IgG. One-way ANOVA was used for statistics in h . All other statistics were performed using students T-test and bar graphs are mean +/− SEM.

Article Snippet: Recombinant Fndc5, Lrg1, IL-15, VEGFβ and TIMP4 were from ABNOVA (Taiwan).

Techniques: Injection, Expressing, Western Blot, Immunohistochemistry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay