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Will R, Bauer K, Kudla M, et al. A Dual HiBiT-GFP-LC3 Lentiviral Reporter for Autophagy Flux Assessment. Methods Mol Biol. 2022;2445:75-98doi: 10.1007/978-1-0716-2071-7_6.
Will, R., Bauer, K., Kudla, M., Montero-Vergara, J., Wiemann, S., Jendrossek, V., Peña-Llopis, S., & Vega-Rubín-de-Celis, S. (2022). A Dual HiBiT-GFP-LC3 Lentiviral Reporter for Autophagy Flux Assessment. Methods in molecular biology (Clifton, N.J.), 244575-98. https://doi.org/10.1007/978-1-0716-2071-7_6
Will, Rainer, et al. "A Dual HiBiT-GFP-LC3 Lentiviral Reporter for Autophagy Flux Assessment." Methods in molecular biology (Clifton, N.J.) vol. 2445 (2022): 75-98. doi: https://doi.org/10.1007/978-1-0716-2071-7_6
Will R, Bauer K, Kudla M, Montero-Vergara J, Wiemann S, Jendrossek V, Peña-Llopis S, Vega-Rubín-de-Celis S. A Dual HiBiT-GFP-LC3 Lentiviral Reporter for Autophagy Flux Assessment. Methods Mol Biol. 2022;2445:75-98. doi: 10.1007/978-1-0716-2071-7_6. PMID: 34972987.
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Methods Mol Biol. 2022;2445:75-98. doi: 10.1007/978-1-0716-2071-7_6.

A Dual HiBiT-GFP-LC3 Lentiviral Reporter for Autophagy Flux Assessment.

Methods in molecular biology (Clifton, N.J.)

Rainer Will, Katja Bauer, Matthias Kudla, Jetsy Montero-Vergara, Stefan Wiemann, Verena Jendrossek, Samuel Peña-Llopis, Silvia Vega-Rubín-de-Celis

Affiliations

  1. Cellular Tools, Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany. [email protected].
  2. Cellular Tools, Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany.
  3. Institute for Cell Biology, University Hospital Essen, Essen, Germany.
  4. Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
  5. Translational Genomics in Solid Tumors, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ) at the University Hospital Essen, Essen, Germany.
  6. Institute for Cell Biology, University Hospital Essen, Essen, Germany. [email protected].

PMID: 34972987 DOI: 10.1007/978-1-0716-2071-7_6

Abstract

Autophagy is an intracellular degradation process that maintains the cellular homeostasis and it is regulated in multiple ways, both in health and disease. Assessment of autophagic flux in cells is an important approach for understanding the function of autophagy in biological contexts. Here, we describe a new tool for the qualitative and quantitative determination of autophagic flux using a dual lentiviral reporter system that generates a fusion HiBiT-GFP-LC3B protein suitable for generating stable cell lines.

© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords: Autophagy; HiBiT-GFP-LC3; Lentivirus

References

  1. Mizushima N, Levine B (2020) Autophagy in human diseases. N Engl J Med 383(16):1564–1576 - PubMed
  2. Mizushima N (2020) The ATG conjugation systems in autophagy. Curr Opin Cell Biol 63:1–10 - PubMed
  3. Vega-Rubin-de-Celis, S.#, and Wiemann, S. Autophagy LC3 HiBiT reporter assay demonstrates mTORC1 regulation of autophagic flux (2018). Promega Reports. https://www.promega.de/en/resources/pubhub/2018/tpub-204-autophagy-lc3-hibit-report-assaysystem- demonstrates-mtorc1-regulation-of-autophagic-flux. - PubMed
  4. Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10(11):1788–1795 - PubMed
  5. Collaboration OR (2016) The ORFeome Collaboration: a genome-scale human ORF-clone resource. Nat Methods 13(3):191–192 - PubMed
  6. Vega-Rubin-de-Celis S, Abdallah Z, Kinch L, Grishin NV, Brugarolas J, Zhang X (2010) Structural analysis and functional implications of the negative mTORC1 regulator REDD1. Biochemistry 49(11):2491–2501 - PubMed
  7. Vega-Rubin-de-Celis S, Kinch L, Peña-Llopis S (2020) Regulation of Beclin 1-mediated autophagy by oncogenic tyrosine kinases. Int J Mol Sci 21(23):9210 - PubMed
  8. Wei Y, Zou Z, Becker N, Anderson M, Sumpter R, Xiao G et al (2013) EGFR-mediated Beclin 1 phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance. Cell 154(6):1269–1284 - PubMed
  9. Kozak M (1999) Initiation of translation in prokaryotes and eukaryotes. Gene 234(2):187–208 - PubMed

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