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Determination of Autophagy in the Caco-2 Spontaneously Differentiating Model of Intestinal Epithelial Cells

  • Sinem Tunçer
  • Sreeparna Banerjee
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1854)

Abstract

The Caco-2 colorectal cancer cell line is widely used as a model for intestinal differentiation and barrier function. These cells, upon reaching confluency, spontaneously differentiate into enterocyte-like cells, synthesize intestinal enzymes, and form domes. Caco-2 cells also undergo autophagy in the course of differentiation. The criteria to establish the induction of autophagy in cells are already well established. Here, we describe the protocol for the spontaneous differentiation of Caco-2 cells and the detection of autophagy using Western blot, flow cytometry, and immunofluorescence.

Keywords

Autophagy Caco-2 Colon Spontaneous differentiation 

Notes

Acknowledgements

This work was funded from TÜBİTAK Project no. 114S937 to S.B. Aslı Sade Memişoğlu, Orkun Cevheroglu, and members of the Banerjee lab are acknowledged for useful discussions. Cagdas Son and Mayda Gursel are acknowledged for sharing resources.

References

  1. 1.
    Tsapras P, Nezis IP (2017) Caspase involvement in autophagy. Cell Death Differ 1–11. doi: 10.1038/cdd.2017.43
  2. 2.
    Klionsky DJ (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717–1721. doi: 10.1126/science.290.5497.1717 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kang R, Zeh HJ, Lotze MT et al (2011) The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 18:571–580. doi: 10.1038/cdd.2010.191 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Wild P, McEwan DG, Dikic I (2014) The LC3 interactome at a glance. J Cell Sci 127:3–9. doi: 10.1242/jcs.140426 CrossRefPubMedGoogle Scholar
  5. 5.
    Slobodkin MR, Elazar Z (2013) The Atg8 family: multifunctional ubiquitin-like key regulators of autophagy. Essays Biochem 55:51–64. doi: 10.1042/bse0550051 CrossRefPubMedGoogle Scholar
  6. 6.
    Kadowaki M, Karim MR (2009) Cytosolic LC3 ratio as a quantitative index of macroautophagy. Methods Enzymol 451:199–213. doi: 10.1016/S0076-6879(08)03613-6 CrossRefGoogle Scholar
  7. 7.
    Karim MR, Kanazawa T, Daigaku Y et al (2007) Cytosolic LC3 ratio as a sensitive index of macroautophagy in isolated rat hepatocytes and H4-II-E cells. Autophagy 3:553–560. doi: 10.4161/auto.4615 CrossRefPubMedGoogle Scholar
  8. 8.
    Katsuragi Y, Ichimura Y, Komatsu M (2015) P62/SQSTM1 functions as a signaling hub and an autophagy adaptor. FEBS J 282:4672–4678. doi: 10.1111/febs.13540 CrossRefPubMedGoogle Scholar
  9. 9.
    Klionsky DJ, Abdalla FC, Abeliovich H et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445–544. doi: 10.4161/auto.19496 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zhang X, Chen S, Huang K et al (2013) Why should autophagic flux be assessed? Acta Pharmacol Sin 34:595–599. doi: 10.1038/aps.2012.184 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326. doi: 10.1016/j.cell.2010.01.028 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Guo S, Liang Y, Murphy SF et al (2015) A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications. Autophagy 11:560–572. doi: 10.1080/15548627.2015.1017181 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Simons BD, Clevers H (2011) Stem cell self-renewal in intestinal crypt. Exp Cell Res 317:2719–2724. doi: 10.1016/j.yexcr.2011.07.010 CrossRefPubMedGoogle Scholar
  14. 14.
    Mizushima N, Levine B (2010) Autophagy in mammalian development and differentiation. Nat Cell Biol 12:823–830. doi: 10.1038/ncb0910-823 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Sambuy Y, De Angelis I, Ranaldi G et al (2005) The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol 21:1–26. doi: 10.1007/s10565-005-0085-6 CrossRefPubMedGoogle Scholar
  16. 16.
    Sun H, Chow EC, Liu S et al (2008) The Caco-2 cell monolayer: usefulness and limitations. Expert Opin Drug Metab Toxicol 4:395–411. doi: 10.1517/17425255.4.4.395 CrossRefPubMedGoogle Scholar
  17. 17.
    Van Beers EH, Al RH, Rings EH (1995) Lactase and sucrase-isomaltase gene expression during Caco-2 cell differentiation. Biochem J 308:769–775. doi: 10.1042/bj3080769 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Buhrke T, Lengler I, Lampen A (2011) Analysis of proteomic changes induced upon cellular differentiation of the human intestinal cell line Caco-2. Dev Growth Differ 53:411–426. doi: 10.1111/j.1440-169X.2011.01258.x CrossRefPubMedGoogle Scholar
  19. 19.
    Dydensborg AB, Herring E, Auclair J et al (2005) Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon. Am J Physiol Gastrointest Liver Physiol 290:G1067–G1074CrossRefGoogle Scholar
  20. 20.
    Pfaffl MW (2001)A new mathematical modelfor relative quantification in real-time RT-PCR. Nucleic Acids Res 29:45eGoogle Scholar
  21. 21.
    Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326. doi: 10.1016/j.cell.2010.01.028 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biological SciencesMiddle East Technical UniversityAnkaraTurkey

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