Category: Assay Development and Screening
Autophagy is an important process cells use for degrading subcellular components in both normal states and diseased states such as cancer where autophagy modulators are expected to have therapeutic potential. To facilitate screening for autophagy modulators, we developed a homogenous cell-based assay using the NanoLuc® Binary Technology (NanoBiT™) that is based on a two-subunit luciferase. The assay incorporates a protein reporter of autophagy comprised of an 11 amino acid NanoBiT subunit (HiBiT) fused by way of a spacer sequence to the N-terminus of the widely used autophagy marker protein LC3. Whereas LC3-based autophagy assays typically require image analysis, we set out to configure an assay that could be analyzed with a simple plate reader. To that end a cDNA encoding our autophagy reporter is expressed in mammalian cells grown in multi-well plates as 2D monolayers or 3D spheroids and detected on a plate reading luminometer. Signals are generated by adding a lytic reagent that contains a luciferase substrate and Large BiT (LgBiT), the NanoBiT subunit that binds with high affinity to the HiBiT segment of the reporter to produce active luciferase and light in proportion to the amount of reporter present. Like native LC3, the LC3-based reporter is degraded by the autophagic pathway and treatments that modulate the rate of autophagic flux are seen as changes in reporter levels. Studies were performed with U2OS and HEK293 cells expressing the reporter from a stably integrated cDNA. The mTOR inhibitors rapamycin, PP242 and AZD8055 caused significant signal decreases, consistent with stimulated autophagic flux and enhanced reporter degradation. In contrast, the autophagy inhibitors bafilomycin A1 and chloroquine significantly enhanced basal signals, consistent with the blockade of autophagic flux and reporter accumulation. The validity of our LC3-based construct as an autophagy reporter was further confirmed by co-treatment with autophagy inducers and bafilomycin A1, which blocked enhanced degradation by the inducers. The magnitude of modulator effects was dose and time dependent and varied somewhat between cell types. Similar results were obtained when the reporter was transiently expressed via simple, one-step transduction with BacMam viral particles. By adding a fluorescent cell necrosis probe to the autophagy assay, it was possible to make same-well determinations of cytotoxicities that might undermine analysis of a compound’s effect on autophagic flux. When assayed in 384-well plates with automation, U2OS and HEK293 stable autophagy reporter cells produced Z’ values of 0.6-0.7 in response to autophagy induction (PP242), and Z’ values of 0.7-0.8 by subsequent autophagy blockade (PP242 + bafilomycin A1). Therefore, using this novel plate-based assay, it is possible to screen test agents and quantitatively assess their effects on autophagic flux in cell culture models.
James Cali– Director, Research, Promega Corporation, Madison, WI
James J. Cali, Ph.D., Director of Research, Promega Corporation, Madison, Wisconsin, USA. Dr. Cali directs Promega's Division of Assay Design. The group uses optical chemistries to provide innovative biological reagents and integrated systems for research and applied technology.