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Assessment of Mitochondrial Dysfunctions After Sirtuin Inhibition

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HDAC/HAT Function Assessment and Inhibitor Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2589))

Abstract

Posttranslational modifications are important for protein functions and cellular signaling pathways. The acetylation of lysine residues is catalyzed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), with the latter being grouped into four phylogenetic classes. The class III of the HDAC family, the sirtuins (SIRTs), contributes to gene expression, genomic stability, cell metabolism, and tumorigenesis. Thus, several specific SIRT inhibitors (SIRTi) have been developed to target cancer cell proliferation. Here we provide an overview of methods to study SIRT-dependent cell metabolism and mitochondrial functionality. The chapter describes metabolic flux analysis using Seahorse analyzers, methods for normalization of Seahorse data, flow cytometry and fluorescence microscopy to determine the mitochondrial membrane potential, mitochondrial content per cell and mitochondrial network structures, and Western blot analysis to measure mitochondrial proteins.

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Acknowledgments

We like to thank Prof. Dr. R. Heller (CMB, University Hospital Jena) for providing HUVEC cells and Dr. Daniel Gebhard (Product Specialist Cell Analysis, Agilent Technologies) for commenting the manuscript. We like to thank the FLI core facilities imaging, flow cytometry, and functional genomics, namely, Dr. Torsten Kroll, who set up the algorithms for automated high-content microscopy analysis, and the mouse facility for their excellent services. The Graduate Academy (GA) of the Friedrich-Schiller-University (FSU) Jena funded L.M-B.

Author information

Authors and Affiliations

  1. Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany

    Christian Marx & Zhao-Qi Wang

  2. Department of Pediatric Hematology and Oncology, Children’s Clinic, Jena University Hospital, Jena, Germany

    Lisa Marx-Blümel & Jürgen Sonnemann

  3. Research Center Lobeda, Jena University Hospital, Jena, Germany

    Lisa Marx-Blümel & Jürgen Sonnemann

  4. Faculty of Biological Sciences, Friedrich-Schiller-University of Jena, Jena, Germany

    Zhao-Qi Wang

Authors
  1. Christian Marx
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  2. Lisa Marx-Blümel
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  3. Jürgen Sonnemann
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  4. Zhao-Qi Wang
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Editor information

Editors and Affiliations

  1. Institute of Toxicology, University Medical Center Mainz, Mainz, Germany

    Oliver H. Krämer

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Marx, C., Marx-Blümel, L., Sonnemann, J., Wang, ZQ. (2023). Assessment of Mitochondrial Dysfunctions After Sirtuin Inhibition. In: Krämer, O.H. (eds) HDAC/HAT Function Assessment and Inhibitor Development. Methods in Molecular Biology, vol 2589. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2788-4_18

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  • DOI: https://doi.org/10.1007/978-1-0716-2788-4_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2787-7

  • Online ISBN: 978-1-0716-2788-4

  • eBook Packages: Springer Protocols

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