Abstract
Endoplasmic reticulum (ER) stress is one of the major mechanisms underlying the etiology of multiple diseases and drug-induced toxicity. Gaussia luciferase (Gluc) is a naturally secreted protein that has been used as a reporter for the secretory pathway of ER to enable efficient and real-time monitoring of the ER function. The Gluc assay has been widely used and optimized in various labs. In this chapter, we provide an example of the application of the Gluc assay by establishing a stable cell line expressing both Gluc and firefly luciferase (Fluc) to study ER stress in liver cells. We describe the detailed procedures used in our laboratory for Gluc- and Fluc-containing lentivirus production and titration, for establishing a HepG2-based stable cell line through lentivirus transduction and the validation process. In addition, we provide an example of using the established stable cell line to investigate ER stress.
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Ellgaard L, Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4(3):181–191. https://doi.org/10.1038/nrm1052
Austin RC (2009) The unfolded protein response in health and disease. Antioxid Redox Signal 11(9):2279–2287. https://doi.org/10.1089/ARS.2009.2686
Urra H, Dufey E, Lisbona F, Rojas-Rivera D, Hetz C (2013) When ER stress reaches a dead end. Biochim Biophys Acta 1833(12):3507–3517. https://doi.org/10.1016/j.bbamcr.2013.07.024
Walter P, Ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334(6059):1081–1086. https://doi.org/10.1126/science.1209038
Xu C, Bailly-Maitre B, Reed JC (2005) Endoplasmic reticulum stress: cell life and death decisions. J Clin Invest 115(10):2656–2664. https://doi.org/10.1172/JCI26373
Uzi D, Barda L, Scaiewicz V et al (2013) CHOP is a critical regulator of acetaminophen-induced hepatotoxicity. J Hepatol 59(3):495–503. https://doi.org/10.1016/j.jhep.2013.04.024
Chen S, Xuan J, Couch L, Iyer A, Wu Y, Li QZ, Guo L (2014) Sertraline induces endoplasmic reticulum stress in hepatic cells. Toxicology 322:78–88. https://doi.org/10.1016/j.tox.2014.05.007
Chen S, Melchior WB Jr, Guo L (2014) Endoplasmic reticulum stress in drug- and environmental toxicant-induced liver toxicity. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 32(1):83–104. https://doi.org/10.1080/10590501.2014.881648
Tannous BA, Kim DE, Fernandez JL, Weissleder R, Breakefield XO (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther 11(3):435–443. https://doi.org/10.1016/j.ymthe.2004.10.016
Verhaegent M, Christopoulos TK (2002) Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal Chem 74(17):4378–4385
Badr CE, Hewett JW, Breakefield XO, Tannous BA (2007) A highly sensitive assay for monitoring the secretory pathway and ER stress. PLoS One 2(6):e571. https://doi.org/10.1371/journal.pone.0000571
Tannous BA (2009) Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat Protoc 4(4):582–591. https://doi.org/10.1038/nprot.2009.28
Nery FC, Armata IA, Farley JE et al (2011) TorsinA participates in endoplasmic reticulum-associated degradation. Nat Commun 2:393. https://doi.org/10.1038/ncomms1383
Maguire CA, Deliolanis NC, Pike L, Niers JM, Tjon-Kon-Fat LA, Sena-Esteves M, Tannous BA (2009) Gaussia luciferase variant for high-throughput functional screening applications. Anal Chem 81(16):7102–7106. https://doi.org/10.1021/ac901234r
Griesenbach U, Vicente CC, Roberts MJ et al (2011) Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer. Biomaterials 32(10):2614–2624. https://doi.org/10.1016/j.biomaterials.2010.12.001
Badr CE, Wurdinger T, Tannous BA (2011) Functional drug screening assay reveals potential glioma therapeutics. Assay Drug Dev Technol 9(3):281–289. https://doi.org/10.1089/adt.2010.0324
Chen S, Zhang Z, Wu Y et al (2015) Endoplasmic reticulum stress and store-operated calcium entry contribute to usnic acid-induced toxicity in hepatic cells. Toxicol Sci 146(1):116–126. https://doi.org/10.1093/toxsci/kfv075
Ren Z, Chen S, Qing T et al (2017) Endoplasmic reticulum stress and MAPK signaling pathway activation underlie leflunomide-induced toxicity in HepG2 cells. Toxicology 392:11–21. https://doi.org/10.1016/j.tox.2017.10.002
Ren Z, Chen S, Zhang J, Doshi U, Li AP, Guo L (2016) Endoplasmic reticulum stress induction and ERK1/2 activation contribute to nefazodone-induced toxicity in hepatic cells. Toxicol Sci. https://doi.org/10.1093/toxsci/kfw173
Ren Z, Chen S, Ning B, Guo L (2018) Use of liver-derived cell lines for the study of drug-induced liver injury. In: Chen M, Will Y (eds) Drug-induced liver toxicity. Methods in pharmacology and toxicology. Humana Press, New York, NY, pp 151–177
Wisniewski JR, Vildhede A, Noren A, Artursson P (2016) In-depth quantitative analysis and comparison of the human hepatocyte and hepatoma cell line HepG2 proteomes. J Proteome 136:234–247. https://doi.org/10.1016/j.jprot.2016.01.016
Bale SS, Vernetti L, Senutovitch N et al (2014) In vitro platforms for evaluating liver toxicity. Exp Biol Med (Maywood) 239(9):1180–1191. https://doi.org/10.1177/1535370214531872
Donato MT, Jover R, Gomez-Lechon MJ (2013) Hepatic cell lines for drug hepatotoxicity testing: limitations and strategies to upgrade their metabolic competence by gene engineering. Curr Drug Metab 14(9):946–968
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Ren, Z., Chen, S., Guo, L. (2020). Methods for Establishing and Using a Stable Cell Line Expressing Both Gaussia Luciferase and Firefly Luciferase to Screen for Endoplasmic Reticulum Stress. In: Keohavong, P., Singh, K., Gao, W. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 2102. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0223-2_29
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DOI: https://doi.org/10.1007/978-1-0716-0223-2_29
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