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Pharmacological and nutritional modulation of autophagy in a rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1.
In Vitro Cellular & Developmental Biology - Animal ( IF 2.1 ) Pub Date : 2020-09-08 , DOI: 10.1007/s11626-020-00490-1
Juan-Ting Liu 1 , Jaramar Balmori-Cedeno 1, 2 , Ehab Misk 1, 3 , John S Lumsden 1
Affiliation  

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.



中文翻译:

虹鳟(Oncorhynchus mykiss)ill细胞系RTgill-W1自噬的药理和营养调节。

自噬参与人类和动物营养,免疫力和疾病的调节。为了了解自噬调节对虹鳟鱼g细胞系RTgill-W1的影响,测试了包括减少营养(2%的胎牛血清与10%的对照组相比),雷帕霉素,3-甲基腺嘌呤,脱氧雪茄烯醇和氯喹的治疗方法。Western blot和免疫荧光检测微管相关蛋白1A / 1B-轻链蛋白,定量聚合酶链反应检测10种自噬相关基因的表达。在治疗后3天,营养显着降低(p  <0.05),自噬显着增加,而脱氧雪腐酚显着(p <0.01)与对照组相比抑制了它。通过使用免疫荧光检测RTgill-W1中自噬体的数量可以确认这些现象。氯喹对于在此模型中观察微管相关蛋白1A / 1B-轻链蛋白至关重要。正如文献所期望的,常用的自噬调节化学物质雷帕霉素和3-甲基腺嘌呤分别激活或抑制了微管相关蛋白1A / 1B-轻链蛋白,但并未始终发挥重要作用。表达10个Atg基因中的5个,包括lc3gabarapatg4atg7atg12与微管相关蛋白1A / 1B轻链蛋白的变化方式相似。自噬相关基因上调的持续趋势,包括用3-甲基腺嘌呤和氯喹处理后的becn1lc3gabarapatg9,提示自噬机制中存在新的反馈调节。

更新日期:2020-09-08
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