Department of Pathobiology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
MOWI West Canada, Campbell River, BC, V9W 8C9, Canada.
In Vitro Cell Dev Biol Anim. 2020 Sep;56(8):659-669. doi: 10.1007/s11626-020-00490-1. Epub 2020 Sep 8.
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 microtubule-associated 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.
自噬参与人类和动物的营养、免疫和疾病的调节。为了了解自噬调节对虹鳟鱼鳃细胞系 RTgill-W1 的影响，我们测试了包括低营养（2%胎牛血清与 10%对照相比）、雷帕霉素、3-甲基腺嘌呤、脱氧雪腐镰刀菌烯醇和氯喹在内的处理方法。使用 Western blot 和免疫荧光检测微管相关蛋白 1A/1B-轻链蛋白，并用定量聚合酶链反应检测 10 个自噬相关基因的表达。在处理后 3 天，与对照组相比，低营养显著（p<0.05）增加了自噬，而脱氧雪腐镰刀菌烯醇显著（p<0.01）抑制了自噬。这些现象通过使用免疫荧光检测 RTgill-W1 中自噬体的数量得到了证实。氯喹对于在该模型中观察微管相关蛋白 1A/1B-轻链蛋白是至关重要的。常用的自噬调节剂雷帕霉素和 3-甲基腺嘌呤分别激活或抑制微管相关蛋白 1A/1B-轻链蛋白，这与文献中的预期一致，但没有一致地显著作用。10 个 Atg 基因中的 5 个，包括 lc3、gabarap、atg4、atg7 和 atg12 的表达模式与微管相关蛋白 1A/1B-轻链蛋白相似。3-甲基腺嘌呤和氯喹处理后自噬相关基因包括 becn1、lc3、gabarap 和 atg9 的上调一致趋势表明自噬机制中存在新的反馈调节。
