Back to Search
Start Over
Monitoring autophagic flux using p62/SQSTM1 based luciferase reporters in glioma cells
- Source :
- Experimental Cell Research. 363:84-94
- Publication Year :
- 2018
- Publisher :
- Elsevier BV, 2018.
-
Abstract
- 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 (p62ΔU) and transfected into cells. The expressed Luc2p-p62 fusion protein was primarily degraded via autophagy, while Luc2p-p62ΔU 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-p62ΔU was determined and the ratio of Luc2p-p62ΔU/Luc2p-p62 was used to assay the autophagic flux. By this approach, the induction of autophagy was manifested as an increased Luc2p-p62ΔU/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-LC3ΔG 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.
- Subjects :
- 0301 basic medicine
Gene knockdown
Cell Survival
ATG5
Autophagy
RNA-Binding Proteins
Apoptosis
Glioma
Cell Biology
Transfection
Biology
Fusion protein
Cell biology
03 medical and health sciences
030104 developmental biology
Cell Line, Tumor
Sequestosome-1 Protein
Humans
Luciferase
Luciferases
Flux (metabolism)
Adaptor Proteins, Signal Transducing
Subjects
Details
- ISSN :
- 00144827
- Volume :
- 363
- Database :
- OpenAIRE
- Journal :
- Experimental Cell Research
- Accession number :
- edsair.doi.dedup.....c726ef94172b23f3dc0162fbcb8ddeeb