Skip to main content
SpringerLink
Log in

Methods for Studying TNFα-Induced Autophagy

  • Protocol
  • First Online:
Immune Mediators in Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2108))

Abstract

Autophagy is an evolutionarily conserved cellular mechanism in eukaryotes that plays an important role in the maintenance of cellular homeostasis. The autophagy process maintains protein homeostasis by recycling damaged organelles and degrading many long-lived proteins in conjunction with the ubiquitin-proteasome system. Cytokines are low-molecular-weight secreted proteins that regulate a broad range of biological activities. For instance, pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα) induce inflammation, autophagy, and apoptotic cell death. In this chapter, we discuss experimental techniques such as immunoblotting and fluorescence microscopy that can be utilized to measure autophagy in response to TNFα treatment.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Chu L-Y, Hsueh Y-C, Cheng H-L, Wu KK (2017) Cytokine-induced autophagy promotes long-term VCAM-1 but not ICAM-1 expression by degrading late-phase IκBα. Sci Rep 7:12472. https://doi.org/10.1038/s41598-017-12641-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Carswell EA, Old LJ, Kassel RL et al (1975) An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A 72:3666–3670. https://doi.org/10.1073/pnas.72.9.3666

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sedger LM, McDermott MF (2014) TNF and TNF-receptors: from mediators of cell death and inflammation to therapeutic giants - past, present and future. Cytokine Growth Factor Rev 25:453–472. https://doi.org/10.1016/j.cytogfr.2014.07.016

    Article  CAS  PubMed  Google Scholar 

  4. Ravikumar B, Sarkar S, Davies JE et al (2010) Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90:1383–1435. https://doi.org/10.1152/physrev.00030.2009

    Article  CAS  PubMed  Google Scholar 

  5. Ryter SW, Cloonan SM, Choi AMK (2013) Autophagy: a critical regulator of cellular metabolism and homeostasis. Mol Cells 36:7–16. https://doi.org/10.1007/s10059-013-0140-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Eskelinen E-L, Saftig P (2009) Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta 1793:664–673. https://doi.org/10.1016/j.bbamcr.2008.07.014

    Article  CAS  PubMed  Google Scholar 

  7. Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728–741. https://doi.org/10.1016/j.cell.2011.10.026

    Article  CAS  PubMed  Google Scholar 

  8. Yoshii SR, Mizushima N (2017) Monitoring and measuring autophagy. Int J Mol Sci 18:1865. https://doi.org/10.3390/ijms18091865

    Article  CAS  PubMed Central  Google Scholar 

  9. Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451:1069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Longatti A, Tooze SA (2009) Vesicular trafficking and autophagosome formation. Cell Death Differ 16:956–965. https://doi.org/10.1038/cdd.2009.39

    Article  CAS  PubMed  Google Scholar 

  11. Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326. https://doi.org/10.1016/j.cell.2010.01.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kabeya Y, Mizushima N, Ueno T et al (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728. https://doi.org/10.1093/emboj/19.21.5720

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E (2005) Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 1:84–91

    Article  CAS  PubMed  Google Scholar 

  14. Rubinsztein DC, Cuervo AM, Ravikumar B et al (2009) In search of an autophagomometer. Autophagy 5:585–589. https://doi.org/10.4161/auto.5.5.8823

    Article  CAS  PubMed  Google Scholar 

  15. Lamark T, Kirkin V, Dikic I, Johansen T (2009) NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets. Cell Cycle 8:1986–1990. https://doi.org/10.4161/cc.8.13.8892

    Article  CAS  PubMed  Google Scholar 

  16. Bjorkoy G, Lamark T, Brech A et al (2005) p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 171:603–614. https://doi.org/10.1083/jcb.200507002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Myeku N, Figueiredo-Pereira ME (2011) Dynamics of the degradation of ubiquitinated proteins by proteasomes and autophagy: association with sequestosome 1/p62. J Biol Chem 286:22426–22440. https://doi.org/10.1074/jbc.M110.149252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Puissant A, Fenouille N, Auberger P (2012) When autophagy meets cancer through p62/SQSTM1. Am J Cancer Res 2:397–413

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Liu WJ, Ye L, Huang WF et al (2016) p62 links the autophagy pathway and the ubiquitin-proteasome system upon ubiquitinated protein degradation. Cell Mol Biol Lett 21:29. https://doi.org/10.1186/s11658-016-0031-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43:67–93. https://doi.org/10.1146/annurev-genet-102808-114910

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Klionsky DJ, Abeliovich H, Agostinis P et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175

    Article  CAS  PubMed  Google Scholar 

  22. Krieg S, Lüscher B, Vervoorts J, Dohmen M (2018) Studying the role of AMPK in autophagy. In: Neumann D, Viollet B (eds) AMPK: methods and protocols. Springer New York, New York, NY, pp 373–391

    Chapter  Google Scholar 

  23. Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798–4811. https://doi.org/10.1038/sj.onc.1209608

    Article  CAS  PubMed  Google Scholar 

  24. Ouyang L, Shi Z, Zhao S et al (2012) Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif 45:487–498. https://doi.org/10.1111/j.1365-2184.2012.00845.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Walker JM (1984) Gradient SDS polyacrylamide gel electrophoresis. Methods Mol Biol 1:57–61. https://doi.org/10.1385/0-89603-062-8:57

    Article  CAS  PubMed  Google Scholar 

  26. Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3:542–545

    Article  CAS  PubMed  Google Scholar 

  27. Jiang P, Mizushima N (2015) LC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells. Methods 75:13–18. https://doi.org/10.1016/j.ymeth.2014.11.021

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Research reported in this chapter was supported by an award from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC2GM125550 to VVD and by the College of Pharmacy and Health Sciences, St. John’s University startup fund to VVD.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, USA

    Sheyda Najafi, Ehab M. Abo-Ali & Vikas V. Dukhande

Authors
  1. Sheyda Najafi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ehab M. Abo-Ali
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vikas V. Dukhande
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vikas V. Dukhande .

Editor information

Editors and Affiliations

  1. Department of Biological Sciences, St. John’s University, Queens, NY, USA

    Ivana Vancurova

  2. Department of Biological Sciences, St. John’s University, Queens, NY, USA

    Yan Zhu

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Najafi, S., Abo-Ali, E.M., Dukhande, V.V. (2020). Methods for Studying TNFα-Induced Autophagy. In: Vancurova, I., Zhu, Y. (eds) Immune Mediators in Cancer. Methods in Molecular Biology, vol 2108. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0247-8_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0247-8_12

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0246-1

  • Online ISBN: 978-1-0716-0247-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation