Objective

Autophagy is a natural process by which cells disassemble unnecessary or malfunctional cellular components for recycling or removal. Dysfunctions of autophagy are thought to be involved in a number of diseases including cancer and neurodegenerative diseases. In this poster we have evaluated a cell line expressing tagged LC3B, a protein widely used as a marker of autophagic “flux”. In this autophagy reporter human LC3B is tagged with HiBiT (11 amino acid peptide) possessing high affinity (Kd~1nM) for LgBiT.  LgBiT and HiBiT are subunits of NanoBiT^TM luciferase (Promega). This is a protein fragment complementation assay (PCFA) that is suitable for the discrimination of both autophagy inhibitors and inducers. Inhibition of autophagy by chloroquine and bafilomycin elevate LC3B levels resulting in an increase in the luminescence signal, while inducers such as the mTOR inhibitors AZD8055 and PP242 decrease LC3B levels with subsequent decrease in luminescence. To validate the response to these compounds within this cell line, we examined the pharmacology of these compounds using the luciferase readout together with other readouts including high content imaging, high throughput Western blotting using Wes^TM and an AlphaLISA^TM assay designed to detect LC3B protein levels in cells. Our data suggest that this assay is suitable for high throughput screening to identify inhibitors or inducers of autophagy.