کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8451482 | 1547695 | 2018 | 35 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Monitoring autophagic flux using p62/SQSTM1 based luciferase reporters in glioma cells
دانلود مقاله + سفارش ترجمه
دانلود مقاله ISI انگلیسی
رایگان برای ایرانیان
کلمات کلیدی
mTORsequestosome 1SQSTM1RAPA3-MAEBSSATG53-methyladenine - 3-متیل آدنینp62/SQSTM1 - P62 / SQSTM1Temozolomide - تموزولامایدRapamycin - راپامایسینAutophagic flux - شار اتوفاژیکDulbecco's phosphate-buffered saline - فسفات باسیل نمک Dulbeccoluciferase - لوسیفرازmicrotubule-associated protein 1 light chain 3 - پروتئین مرتبط با میکروتوبول 1 زنجیره سبک 3Chloroquine - کلروکین Glioma - گلیوما
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
تحقیقات سرطان
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Autophagy is a highly dynamic process characterized with the term of autophagic flux. In the present study, we developed a quantifiable luciferase reporter system to measure the capacity as well as the dynamics of autophagic flux. Briefly, a luciferase variant of Luc2p was fused with p62/SQSTM1 or its UBA domain deletion mutant (p62ÎU) and transfected into cells. The expressed Luc2p-p62 fusion protein was primarily degraded via autophagy, while Luc2p-p62ÎU was employed as a normalization control due to its resistance to autophagic degradation. The luciferase activity of the lysates from two parallel populations of glioma cells expressing either Luc2p-p62 or Luc2p-p62ÎU was determined and the ratio of Luc2p-p62ÎU/Luc2p-p62 was used to assay the autophagic flux. By this approach, the induction of autophagy was manifested as an increased Luc2p-p62ÎU/Luc2p-p62 ratio, which could be neutralized by autophagy inhibitors or knockdown of ATG5. The performance of our autophagic flux detection system was comparable to a recently reported GFP-LC3-RFP-LC3ÎG probe. We tested the system in TMZ treated glioma cells, and found that coadministration of chloroquine to attenuate cellular autophagic flux significantly improved the TMZ efficacy by triggering more early apoptosis. Collectively, our luciferase-based autophagic flux assay may serve as a useful alternative yet sensitive method for autophagic flux detection in tumor cells.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Experimental Cell Research - Volume 363, Issue 1, 1 February 2018, Pages 84-94
Journal: Experimental Cell Research - Volume 363, Issue 1, 1 February 2018, Pages 84-94
نویسندگان
Zhang Min, Yao Ting, Gong Mingtao, Tang Xiaofei, Yan Dong, Zhang Chenguang, Ding Wei,