Autophagy is an evolutionarily conserved catabolic process in which cytosolic content is engulfed, degraded in lysosomes, and recycled. This process is critical to maintain cellular homeostasis, and it has been implicated in numerous diseases, including neurodegenerative diseases, inflammatory diseases, and cancer. Our goal is to discover small-molecule modulators of this pathway to study autophagy in these various disease states. A HeLa cell line stably expressing Green Fluorescent Protein (GFP)-labeled LC3 was used to develop a high-content screen for autophagy modulation. GFP-LC3-I is located diffusely throughout the cytosol, but when autophagy is activated, LC3-I is conjugated to phosphatidylethanolamine to form GFP-LC3-II, which is recruited to the autophagosome membrane, forming GFP-LC3 puncta. These puncta can be counted using fluorescence microscopy to enumerate the number of puncta per cell, which correlates to the number of autophagosomes and indicates the degree to which autophagy is active. Efforts to optimize the assay to enable the identification of both autophagy activators and inhibitors, as well as the preliminary results of the high-content screen, will be presented.

Optimization of a high-content screen for autophagy modulators

254th National Meeting (2017)

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