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Loss-of-Function RNAi Screen to Identify Necrosis-Signaling Molecules

  • David Mark Moquin
  • Francis Ka-Ming ChanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1857)

Abstract

Over the recent years, genome-wide RNA interference (RNAi) library screens have been instrumental in the identification of key regulators of various biological pathways. The prolific use of this technique is attributed to its amenability to a high-throughput format. Here, we present the step-by-step method to conduct a siRNA screen to identify genes involved in necroptosis, a nonapoptotic form of proinflammatory cell death. The method described here uses MTS cell proliferation assay to measure necroptosis, which is compatible with high-throughput format screening on multiwell microtiter plates. This ensures that the screen can be performed in a timely and efficient manner.

Key words

Necroptosis Programmed necrosis Cell death Caspase-independent TNFα Transfection siRNA screen 

References

  1. 1.
    Chan FK, Luz NF, Moriwaki K (2015) Programmed necrosis in the cross talk of cell death and inflammation. Annu Rev Immunol 33:79–106CrossRefPubMedGoogle Scholar
  2. 2.
    Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, Chan FK (2009) Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137:1112–1123CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    He S, Wang L, Miao L, Wang T, Du F, Zhao L, Wang X (2009) Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 137:1100–1111CrossRefPubMedGoogle Scholar
  4. 4.
    Hitomi J, Christofferson DE, Ng A, Yao J, Degterev A, Xavier RJ, Yuan J (2008) Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 135:1311–1323CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Moquin DM, McQuade T, Chan FK (2013) CYLD deubiquitinates RIP1 in the TNFalpha-induced necrosome to facilitate kinase activation and programmed necrosis. PLoS One 8:e76841CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Zhao J, Jitkaew S, Cai Z, Choksi S, Li Q, Luo J, Liu ZG (2012) Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci U S A 109:5322–5327CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wilson RC, Doudna JA (2013) Molecular mechanisms of RNA interference. Annu Rev Biophys 42:217–239CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Chan FK, Shisler J, Bixby JG, Felices M, Zheng L, Appel M, Orenstein J, Moss B, Lenardo MJ (2003) A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses. J Biol Chem 278:51613–51621CrossRefPubMedGoogle Scholar
  9. 9.
    Chan FK, Lenardo MJ (2000) A crucial role for p80 TNF-R2 in amplifying p60 TNF-R1 apoptosis signals in T lymphocytes. Eur J Immunol 30:652–660CrossRefPubMedGoogle Scholar
  10. 10.
    Chan FK, Moriwaki K, De Rosa MJ (2013) Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol Biol 979:65–70CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Wang L, Du F, Wang X (2008) TNF-alpha induces two distinct caspase-8 activation pathways. Cell 133:693–703CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of ImmunologyDuke University Medical CenterDurhamUSA

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