Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux

Autophagy. 2009 Oct;5(7):1018-25. doi: 10.4161/auto.5.7.9443. Epub 2009 Oct 6.


Macroautophagy (hereafter referred to as autophagy) has recently emerged as an attractive target for the treatment of various degenerative diseases and cancer. The discovery of effective pharmaceutical regulators of autophagy has, however, been hindered by a lack of feasible assay systems for autophagic flux. Here, we present a luciferase-based reporter assay that measures autophagic flux in real time in living cells and demonstrate that this assay system is apt for the detection of dose- and stimulus-dependent differences in autophagy kinetics. Furthermore, by screening a small molecule kinase inhibitor library containing 80 compounds we identified 12 compounds as inducers of autophagic flux. Importantly, six inhibitors of the class I phosphoinositide 3-kinase -- protein kinase B -- mammalian target of rapamycin complex 1 axis, the central signaling pathway repressing autophagy, scored as autophagy inducers adequately validating the screen. We conclude that the assay system presented here allows easy and rapid monitoring of autophagy kinetics and is suitable for screening of small molecule libraries.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anti-Bacterial Agents / metabolism
  • Antineoplastic Agents, Phytogenic / metabolism
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism
  • Autophagy / physiology*
  • Beclin-1
  • Biological Assay / methods*
  • Cells, Cultured
  • Enzyme Inhibitors / metabolism
  • Etoposide / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / physiology
  • Genes, Reporter
  • Humans
  • Luciferases / genetics
  • Luciferases / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mice
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • RNA, Small Interfering / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Sirolimus / metabolism


  • Anti-Bacterial Agents
  • Antineoplastic Agents, Phytogenic
  • Apoptosis Regulatory Proteins
  • BECN1 protein, human
  • Beclin-1
  • Enzyme Inhibitors
  • LC3 protein, rat
  • Membrane Proteins
  • Microtubule-Associated Proteins
  • RNA, Small Interfering
  • Recombinant Fusion Proteins
  • Etoposide
  • Luciferases
  • Sirolimus