Related Experiment Video
Updated: May 13, 2026
Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry
Published on: July 21, 2017
Identification of small-molecule autophagy activators via GFP-LC3 high-throughput screening and a cargo-based
Freke Mertens1, Farnaz Sedigheh Takhsha2, Mélissa Lallier3
1Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium; Infla-Med Centre of Excellence, University of Antwerp, Antwerp, Belgium; Peripheral Neuropathy Research Group, University of Antwerp, Antwerp, Belgium.
Background And Aim:
Autophagy maintains cellular homeostasis by recycling macromolecules and nutrients. It involves the sequestration of superfluous or damaged cellular components into autophagosomes, which fuse with lysosomes for degradation. Reduced autophagy is implicated in numerous diseases, which may be treatable with autophagy-inducing drugs. However, most clinically available inducers act through mTORC1 inhibition, causing off-target effects that limit their therapeutic use. This study aimed to identify novel autophagy-inducing compounds that act independently of mTORC1, thereby offering greater translational potential.
Methods:
A high-throughput imaging assay was optimised to quantify autophagosome-like structures in L929 fibroblasts expressing GFP-LC3, a fluorescent autophagosome membrane marker. Hits were validated alongside several reference autophagy modulators in retinal epithelial hTERT RPE-1 cells expressing the LDHB-mKeima autophagy cargo reporter. This assay distinguishes functional autophagy flux inducers from compounds that merely increase autophagosome accumulation by blocking late-stage autophagy. Western blotting was used to investigate the mechanism of autophagy initiation.
Results:
High-throughput screening identified 30 hits that increased autophagosome-like structures more than fourfold. Ten compounds were confirmed to induce autophagic flux of bulk cargo. Nine of these acted independently of mTORC1, while elevating autophagic flux to a similar extent as the mTORC1 inhibitor rapamycin.
Conclusion:
While GFP-LC3-based assays enabled efficient high-throughput screening, incorporation of the LDHB-mKeima cargo-based assay was essential for identifying functional autophagy flux activators. Nine compounds were identified that promoted autophagic cargo flux via mTORC1-independent mechanisms, providing promising leads for discovering new molecular targets and developing safer, more effective autophagy-based interventions to treat human disease.
