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Gene-Protein-metabolism links Cell Metabolism Assays for OMICS Research STANDARD Parameters of Functional METABOLIsm Genomics Cells use gene expression to synthesize proteins and other products that are essential for various cellular functions. Gene expression levels, while associated with metabolism, cannot solely predict the functional impact on metabolism. This creates a gap in understanding the complex physiological relation between genes and metabolism. Researchers are using XF Technology to link functional metabolism to gene expression for a complete picture. The XF Cell Mito Stress Test measures the key parameters of mitochondrial respiration: basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. The XF Glycolysis Stress Test measures the key parameters of glycolytic activity: glycolysis, glycolytic capacity, glycolytic reserve. Oxygen Consumption Rate (OCR) OCR (pmol/min) (pmol/min) 500 Control Hdac9 deleted * 400 300 200 100 FCCP FCCP Mouse T cells pmol O2 per min per µg protein Oligomycin oligomycin Basal basal Basal respiration 4 pmol O2 per min per mg protein 0 P<0.05 3 2 XF Assay reveals a link between histone deacetylase 9 (Hdac9) deletion and mitochondrial 1 respiration correlating to a genetic and functional increase in T regulatory cells. (Beier UH et al., 2015. FASEB J.) 0 L P<0.05 4 2 0 1 iPS S- LA ME 3 iPS S- LA ME 2 iPS S- LA ME iPS S- LA ME Maximum respiration 6 iPS S- iPS Spare 6 Capacity P<0.05 Oxidative reserve 3 4 2 2 1 0 1 4 P<0.05 2 0 P<0.05 3 2 1 3 2 iPS -iPS iPS -iPS iPS SSS S SLA ELA LA LA LA ME ME M ME 1 S1 S33 S22 -iPS -iPS -iPS SiP A-SiP A-SiP LS LSAELS M M M LEA LA LEA ME S1 -iP S LA ME iPS S- LA ME ME ME 1 0 3 S1 S2 PSS33 SS22 S1 iPS i-P -i S- AS -SiP-iP - iP LAS-iPSA-SiP LA ALS LSA A E LEAL L ME EMLE ME EM EM M MELAS-iPS1 2 1 0 10 5 1 iPS S- LA ME 15 MELAS-iPS2 2 1 S3 S1 iPS2 PS3 i -iP S-iP SAS AS 0 MEL ELA MEL ELA M M 0 25 P<0.05 4 2 0 S11 -iPS SiP LSA- ME 3 22 PS3 PS S iP-iS iSP-iS LAS SLAE E A A LM LM OCT4 SOX1 PAX6 10000 1000 NANOG NESTIN 100 10 1 0.1 0.01 0.001 MELAS-IPS1 MELAS-IPS2 MELAS-IPS3 2 50 75 iPS S- LA ME P<0.05 2 1 0 100 Human neuronal progenitor cells XF Cell Mito Stress Test predicts differentiation status in neuronal progenitor cells (NPCs) derived from patients with mutations causing mitochondrial encepharopathy and stroke-like episodes (MELAS) which correlates to gene expression. S1 -iP AS ME 100000 3 iPS S- LA ME 3 L ME (min) TimeTime (min) (Ma H et al., 2015. Nature.) 1 iPS S- Oxidative reserv iPS S- LA ME 0 LA MELAS-iPS3 & MELAS-iPS1 MELAS-iPS2 Rotenone MELAS-iPS3 3 Oligomycin FCCP antimycin RotenoneA& P<0.05 P<0.05 antimycin A Oligomycin FCCP 0 6 E M LA ME M Oxidative reserve Oxidative reserve 3 1 ME pmol O2 per min per µg protein P<0.05 2 M Maximum respiration Maximum respiration 6 E M0 0 S- LA ME ME 1 S2 -iP S LA 2 L ME 2 iPS AS 2 Maximal Respiration Oxygen Consumption Rate (OCR) (pmol/min/µg protein) pmol O2 per min per µg protein 1 Oxygen Consumption Rate (OCR) µg protein pmol O2 per min per protein) (pmol/min/µg 2 LA ME pmol O2 per min per µg protein 1 0 P<0.05 3 P<0.05 3 P<0.05 L ME 3 iPS AS P<0.05 Maximum respiration Relative Expression Levels iP S- LA ME S2 1 3 pmol O2 per min per µg protein iP S- LA ME S3 2 2 pmol O2 per min per µg protein iP S- LA ME S1 P<0.05 ATP turnover 33 0 SiP3S3 PS1 -SiP2S2 -iPS3 SS11 -AiP -iSP-iP S- AS-i S-AiP S S A S S 0EL AEL MEL LMAEL MELA A L L M M 1 2 ME M3E ME Oxygen Consumption Rate (OCR) per min per µg protein pmol O2pmol O2 per min per µg protein (pmol/min/µg protein) 0 4 pmol O2 per min per µg protein 1 4 ATP Production ATP turnover ATP turnover OxygenOxygen Consumption Rate (OCR) Consumption Rate (pmol/min/µg protein) (pmol O2 per µgprotein) 2 Basal respiration Basal respiration pmol O2 per min per mg protein P<0.05 3 Basal Respiration per min permin µg protein pmol O2pmol (pmol/min/mg protein) O2 per per mg protein Basal respiration 4 Oxygen Consumption Rate (OCR) ME 1 iPS AS ATP turnover 3 S3 -iP AS L ME iPS S- LA ME THE WORLD’S MOST ADVANCED METABOLIC ANALYZERS Proven Technology for Cutting Edge Research There are over 2,000 references utilizing XF Technology published in leading journals such as Nature and Cell. Scientists are embracing XF Technology to identify metabolic phenotypes and reprogramming to target metabolic pathways for therapeutic purposes. Glucose Pyruvate Lactate IV III II GLYCOLYSIS — ECAR (Extracellular Acidification Rate) MITOCHONDRIAL RESPIRATION — OCR (Oxygen Consumption Rate) I Cells generate ATP Mitochondria consume via glycolysis independent oxygen when oxidizing fatty of oxygen, producing lactic acid acids or other substrates and protons. XF Analyzers measure to generate ATP. XF glycolysis by measuring the extracel- Analyzers measure mitochondrial lular acidification rate (ECAR) of respiration by measuring the oxygen cells. consumption rate (OCR) of cells. Proteomics Advances in proteomic technology enables genome-wide data analysis, pinpointing specific proteins and expression levels. Proteomic data directly links to metabolism without requiring the regulatory mechanisms associated with mRNA expression and final protein expression. However, conclusions utilizing only these types of experiments are limited. XF Technology not only confirms proteomic data but also allows for further investigation of protein function. WT PKA-deficient 2.0 + 1.50-1.99 0.51-0.70 0.5- ATP-linked Respiration Rate (pmol O2 /min/ 1x105 cells) 150 * 50 0 CPT-cAMP - + - + Murine lymphoma cells M-BMM M-BMM M-BMM GM-BMM Glucose Oligomycin 2-DG Glucose Oligomycin 2-DG 0.71-1.49 Not represented 100 GM-BMM GM-BMM GM-BMM Microarray Fold Change 50 Extracellular Acidification Rate (ECAR) (mpH/min) 200 Glucose Oligomycin 50 40 GO_BP: Gene & Protein Expression Profile M-BMM Glucose metabolic process mRNA mRNA 2-DG RPIA PYGL PFKP LDHB GYS1 FABP5 ENO1 ALDOC 40 30 50 3020 10 20 40 10 0 30 0 20 *** 1 1 30 30 10 0 60 0 0 0 0 90 120 GM-BMM 15 18 21 24 Time Glucose (min)Oligomycin 60 *** M-BMM 2-DG 9500 120 150 180 210 240 40 Time (min) 30 20 *** 10 1 30 0 610 30 0 protein protein 1 2 3 10 relative expression level GM-BMM/ M-BMM 12 *** 9600 90121200 1510 51080 2110 82040 210 240 Time (min) Time (min) Murine bone-derived macrophages XF Cell Mito Stress Test reveals that protein kinase A (PKA)dependent changes in protein and gene expression correlate with changes in ATP-linked respiration. XF Glycolysis Stress Test reveals the increased glycolytic capacity of granulocyte-stimulated macrophages (M-BMM), confirming the inherently higher glycolytic protein expression. (Wilderman A et al., 2015. J Biol Chem.) (Na YR et al., 2015. Mol Cell Proteomics.) MEASURING THE KEY PARAMETERS OF Functional Cell metabolism Metabolomics Techniques, such as mass spectrometry (MS) and nuclear magnetic resonance (NMR), enable the analysis of hundreds of metabolites in a given sample. However, the data from these techniques alone are not sufficient to assess functionality within a biological context. XF Technology provides real-time analysis of cellular response to stress or nutrients, and can direct metabolomics research, adding efficiency and direction to any area of research. ** 0 F6P FBP 1 2 pmol/106 cells 350 3 G3P * ** 350 0 3PG 1 2 3 0 1 2 PEP 3 20,000 pmol/106 cells pmol/106 cells ** 175 175 ATP 60 R5P X5P E4P * 10,000 0 1 2 3 50,000 25,000 0 Lactate ** LDHs PKMs Pyruvate PDHs ** 0 1 2 3 2 3 * 90 45 0 300 1 “With very little learning curve, XF Technology provides highquality data. We generated publishable results within the first month.” *** *** 150 0 1 120 ** 60 2 3 ** 0 1 2 1,000 * 3 500 0 Acetyl-CoA 1 2 3 Medical School 40 3 * 1,250 Ru5P 6PG * 80 pmol/106 cells pmol/106 cells 2 G6P 1 2 3 pmol/106 cells 1 2,500 10 pmol/106 cells 0 100 pmol/106 cells 175 * *** 1,000 pmol/106 cells GLUTs HKs 1 2 3 - Researcher at Case Western PC9 GEF ERLO DMSO GEF ERLO ERLO DMSO 0 GEF Reserve University HCC827 H1975 Human lung adenocarcinoma cells XF Glycolysis Stress Test predicts sensitivity to first-line EGFR inhibitors used in lung adenocarcinoma cells, and correlates to reduced metabolite levels. Extracellular AcidificationRate Rate (ECAR) Extracelluar (ECAR) Extracelluar Acidification Acidification Rate (ECAR) (mpH/min) Extracelluar (mpH/min) Acidification Rate (ECAR) (mpH/min) (Makinoshima H et al., 2014. J Biol Chem.) Th1 Th1 Th1 Th17 Th17 Th17 Th17 Th1 60 60 Treg Treg Treg Treg Glucose Glucose Oligomycin Oligomycin Glucose Oligomycin 2-DG California, San Diego 50 (mpH/min) 40 40 40 30 30 30 1 20 20 Oxygen Consumption Rate (OCR) Oxygen Consumption Rate Oxygen Consumption Rate (OCR) (OCR) Oxygen Consumption Rate (OCR) (pmol/min) (pmol/min) (pmol/min) (pmol/min) 0 G6P/F6P Oxidative PPP Th1 Th17 Treg 6P Gluconate 15 10 00 8 6 4 2 0 1.5 Glucose 0.5 Glycolysis 20 10 5 Relative level normalized to naive (red line) 0 20 15 0 00 1400 1400 1400 25 50 25 25 75 Time (min) 50 50 100 75 75 125 150 100 100 125 125 Rotenone & Oligomycin FCCP antimycin Rotenone Rotenone && A Fructose 10 1,6-P 5 20 15 150 150 Glycerol 3P 5 0 0 2.5 2 1.5 1 0.5 0 0 5 4 3 0 1 5 4 3 2 1 0 Lactate PEP 0.5 0 3 2 Pyruvate 1 0 Sedoheptulose 7P Eryhrose 4P 1 1,3-P Glycerate 0.5 1.5 600 800 800 5 1.5 Glycerate 2 1 3P 25 20 15 10 5 0 15 10 G3P DHAP 10 1200 1000 1000 800 Nonoxidative PPP 0 5 4 3 2 1 0 Oligomycin Oligomycin FCCP FCCP antimycin antimycinAA 1200 1200 1000 2 1.5 1 0.5 0 CisAconitate Citrate 2 1.5 1 0.5 0 5 4 3 2 1 0 6 2.5 2 1.5 1 0.5 0 Succinate 4 2 6 0 Oxaloacetate 4 Acetyl CoA α-Keto glutarate TCA cycle Fumarate 2 0 Malate 400 600 600 200 400 400 0 0 200 200 00 00 25 50 75 Time (min) Time (min) 100 125 Murine T cells 25 25 50 50 75 75 Time Time(min) (min) “When we decided to more closely investigate branched chain amino acid metabolism, we chose to incorporate our XF Technology, knowing that it is a powerful tool for assessing mitochondrial function and metabolism.” - Researcher at University of 2-DG 2-DG 60 50 50 10 100 “We were really surprised by the impact of measuring metabolism in our model system. This opens up new research avenues for us.” - Researcher at Rosalind Franklin 10,000 pmol/106 cells pmol/106 cells Glucose 350 DHAP DMSO Extracellular Acidification Rate (ECAR) ECAR (mpH/min) (mpH/min) ERLO ** *** *** *** 120 GEF pmol/106 cells GEF= Gefitinib DMSO ERLO= Erolotinib What OUR Customers ARE SAYING 150 100 100 125 125 150 150 XF Glycolysis Stress Test and XF Cell Mito Stress Test reveal increased levels of mitochondrial respiration and glycolytic activity in TH17 (T cells) which correlate to changes in metabolite levels. (Gerriets VA et al., 2015. J Clin Invest.) Functional metabolic assayS The Genotype-Phenotype Link The traditional paradigm encompassing the flow of information from gene to protein to function can be found in nearly every biology and biochemistry textbook. At the time, this discovery was thought to explain all of biology, health, and disease. However, only focusing on the expression of a single gene or protein neglects the multi-faceted consequences of that expression on metabolism, as well as overall cell and tissue health. Mitochondria are at the crux of cellular energy generation, intracellular signaling, and cell death and survival regulation. Compromised mitochondria can strain the expansive and intricate cellular metabolic network. There is increasing appreciation for the role of mitochondria in disease pathology, etiology, and cell health. Integrating processes that demonstrate the link amongst genes, proteins, and metabolism allows researchers to analytically answer their scientific questions. XF Technology provides researchers the necessary tools to measure the functional, metabolic impact of gene and protein expression, faster and easier using live cells. GENES & METABOLISM LINK Gene Expression Functional Metabolism Full Picture of Cell Health Protein Expression substrates and metabolism GLUCOSE GLUCOSE PI3/AKT p53 GLYCOLYSIS PYRUVATE Acetyl-CoA TCA LIPID SYNTHESIS GLUTAMATE GLUTAMATE PI3/AKT ETC TCA GLYCOLYSIS (ECAR) MYC GLUTAMINOLYSIS CITRATE PYRUVATE HIF1 MYC FATTY ACID LACTATE MYC HIF1 HIF1 FATTY ACID MITOCHONDRIAL RESPIRATION (OCR) GLUTAMINE Gold Standard metabolic Assays MEASURING THE KEY PARAMETERS OF CELL METABOLISM XF Glycolysis Stress Test Profile XF Cell Mito Stress Test Profile Glycolytic Function Mitochondrial Respiration FCCP Extracellular Acidification Rate (ECAR) (mpH/min) Oxygen Consumption Rate (OCR) (pmol/min) Oligomycin Rotenone & antimycin A 360 320 280 Spare Capacity 240 200 160 120 80 Basal Respiration Maximal Respiration Proton Leak Non-mitochondrial Oxygen Consumption 40 0 ATP Production 0 10 20 30 40 50 60 70 Time (minutes) 80 90 100 110 Glucose Oligomycin 2-DG 45 40 35 Glycolytic Reserve 30 25 Glycolytic Capacity 20 15 Glycolysis 10 5 0 Non-glycolytic Acidification 0 10 20 30 40 50 60 70 Time (minutes) 80 90 100 XF Mito Fuel Flex Test Profile XF Cell Energy Phenotype Test XF Mito Fuel Flex Test Profile Metabolic Phenotype & Potential XFp Cell Energy Phenotype Profile Mitochondrial MitochondrialFunction Function Baseline Phenotype Quiescent Glycolytic Glycolysis Extracellular Acidification Acidification Rate (ECAR) Extracellular Rate (ECAR) 60 Flexibility 40 Flexibility 20 Dependency 0 Glucose Pathway Glucose Pathway Dependency Dependency Glutamine Pathway Fatty Acid Pathway Glutamine Pathway Fatty Acid Pathway Corporate Headquarters European Headquarters Asia-Pacific Headquarters Seahorse Bioscience Inc. 16 Esquire Road North Billerica, MA 01862 US Phone: +1.978.671.1600 www.seahorsebio.com Seahorse Bioscience Europe Fruebjergvej 3 2100 Copenhagen DK Phone: +45 31 36 98 78 Seahorse Bioscience Asia 199 Guo Shou Jing Rd, Suite 207 Pudong, Shanghai 201203 CN Phone: 0086 21 33901768 © 2015 All rights reserved. Seahorse Bioscience and the Seahorse logo are trademarks of Seahorse Bioscience Inc. The content in this brochure is for informational purposes only and may be subject to change without notice. Rev 1 n Capacity te Capacity ab Po 80 Capacity M et ic ol l tia % GLC+GLN+FA % GLC+GLN+FA FuelOxidation Oxidation Stressed Phenotype Mitochondrial Respiration OxygenConsumption Consumption Rate Oxygen Rate(OCR) (OCR) 100 Energetic Aerobic