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Autophagy pp 389-428 | Cite as

In Vitro Screening Platforms for Identifying Autophagy Modulators in Mammalian Cells

  • Elena Seranova
  • Carl Ward
  • Miruna Chipara
  • Tatiana R. Rosenstock
  • Sovan SarkarEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)

Abstract

Autophagy is a vital homeostatic pathway essential for cellular survival and human health. It primarily functions as an intracellular degradation process for the turnover of aggregation-prone proteins and unwanted organelles. Dysregulation of autophagy underlying diverse human diseases reduces cell viability, whereas stimulation of autophagy is cytoprotective in a number of transgenic disease models including neurodegenerative disorders. Thus, therapeutic exploitation of autophagy is considered a potential treatment strategy in certain human diseases, and therefore, chemical inducers of autophagy have tremendous biomedical relevance. In this review, we describe the in vitro screening platforms to identify autophagy modulators in mammalian cells using various methodologies including fluorescence and high-content imaging, flow cytometry, fluorescence and luminescence detection by microplate reader, immunoblotting, and immunofluorescence. The commonly used autophagy reporters in these screening platforms are either based on autophagy marker like LC3 or autophagy substrate such as aggregation-prone proteins or p62/SQSTM1. The reporters and assays for monitoring autophagy are evolving over time to become more sensitive in measuring autophagic flux with the capability of high-throughput applications for drug discovery. Here we highlight these developments and also describe the stringent secondary autophagy assays for characterizing the autophagy modulators arising from the primary screen. Since autophagy is implicated in myriad human physiological and pathological conditions, these technologies will enable identifying novel chemical modulators or genetic regulators of autophagy that will be of biomedical and fundamental importance to human health.

Key words

Autophagy Autophagy substrates Autophagy modulators Autophagy drug discovery LC3 p62 Aggregation-prone protein Chemical screen Screening platform 

Notes

Acknowledgments

We thank the funding agencies for supporting our research. SS is funded by Wellcome Trust Seed Award (109626/Z/15/Z), UKIERI (UK-India Education and Research Initiative) DST Thematic Partnership Award (2016-17-0087), and Birmingham Fellowship. TRR is funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2015/02041-1), Fundação de Apoio à Pesquisa da Faculdade de Ciências Médicas da Santa Casa de São Paulo (FAP FCMSCSP), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). SS is also a Former Fellow for life at Hughes Hall, University of Cambridge, UK.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Elena Seranova
    • 1
  • Carl Ward
    • 1
  • Miruna Chipara
    • 1
  • Tatiana R. Rosenstock
    • 2
  • Sovan Sarkar
    • 1
    Email author
  1. 1.Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
  2. 2.Department of Physiological ScienceSanta Casa de São Paulo School of Medical ScienceSão PauloBrazil

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