Pharmacological and nutritional modulation of autophagy in a rainbow trout ( Oncorhynchus mykiss ) gill cell line, RTgil
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Pharmacological and nutritional modulation of autophagy in a rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1 Juan-Ting Liu 1 & Jaramar Balmori-Cedeno 1,2 & Ehab Misk 1,3 & John S. Lumsden 1 Received: 11 February 2020 / Accepted: 6 August 2020 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2020
Abstract Autophagy is involved in the modulation of nutrition, immunity, and disease in humans and animals. To understand the impact of autophagy modulation on a rainbow trout gill cell line, RTgill-W1, treatments including reduced nutrition (2% fetal bovine serum compared with 10% control), rapamycin, 3-methyladenine, deoxynivalenol, and chloroquine were tested. Western blot and immunofluorescence were used to detect microtubule-associated protein 1A/1B-light chain protein and quantitative polymerase chain reaction was used to detect the expression of 10 autophagy-related genes. At 3-d post-treatment, reduced nutrition significantly (p < 0.05) increased autophagy while deoxynivalenol significantly (p < 0.01) suppressed it compared to controls. These phenomena were confirmed by using immunofluorescence to detect the number of autophagosomes in RTgill-W1. Chloroquine is critical to allow observation of microtubule-associated protein 1A/1B-light chain protein in this model. The commonly used autophagy-modulating chemicals rapamycin and 3-methyladenine either activated or suppressed microtubuleassociated protein 1A/1B-light chain protein, respectively, as expected from the literature, but did not act in a consistently significant manner. Expression of five of the 10 Atg genes, including lc3, gabarap, atg4, atg7, and atg12, were altered in a similar pattern to microtubule-associated protein 1A/1B-light chain protein. The consistent trend of autophagy-related gene upregulation including becn1, lc3, gabarap, and atg9 following treatment with 3-methyladenine and chloroquine is suggestive of a novel feedback regulation in the autophagy machinery. Keywords Autophagy . RTgill-W1 . Deoxynivalenol . Chloroquine . Reduced nutrition . Autophagy-related genes
Introduction Autophagy is a highly conserved mechanism in all eukaryotic cells that responds to negative environmental conditions such
* John S. Lumsden [email protected] Juan-Ting Liu [email protected] Jaramar Balmori-Cedeno [email protected] Ehab Misk [email protected] 1
Department of Pathobiology, University of Guelph, Guelph, ON N1G 2W1, Canada
2
Present address: MOWI West Canada, Campbell River, BC V9W 8C9, Canada
3
Present address: Huntsman Marine Science Center, Saint Andrews, NB E5B 2L7, Canada
as starvation by degrading cytoplasmic structures for recycling of the components (Mortimore and Schworer 1977; Kirisako et al. 1999; Zinke et al. 2002; Yorimitsu and Klionsky 2005). Macroautophagy (hereafter, termed autophagy) activation results in the production of an autophagosome that selectively targets and engulfs cytoplasmic contents (Ichimura et al. 2013; Lim et al. 2015), which are digested in the autophagolysosome. The func
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