Robust LC3B lipidation analysis by precisely adjusting autophagic flux

Abstract

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

Discovery of a Small-Molecule Modulator of the Autophagy-Lysosome Pathway That Targets Lamin A/C and LAMP1, Induces Autophagic Flux, and Affects Lysosome Positioning in Neurons

“…To further confirm the conclusion from the dual reporter assay that 1a and 2a activate autophagy, LC3-II levels were also quantified with western blot flux assays (Figure E). If the compounds were late-stage inhibitors, we would expect to see no additional increase in LC3-II levels with co-treatment of the vATPase inhibitor, BafA1; however, we observed a significant increase in LC3-II levels following treatment with both compounds and a further increase when BafA1 was added to prevent LC3-II turnover through late-stage autophagy inhibition (Figure F). , These results support the conclusion from the dual reporter assay that 1a and 2a are true autophagy activators and are enhancing autophagic flux. Next, we attempted to determine if our hits were activating autophagy independently of mTOR.…”

An AI-guided screen identifies probucol as an enhancer of mitophagy through modulation of lipid droplets

“…A recent report found that autophagy must be tuned to provide sufficient dynamic range to resolve differences in LC3 lipidation following manipulations such as drug treatments [ 32 ]. This can be accomplished by employing bafilomycin, an inhibitor of lysosome–autophagosome fusion at low doses so the effects of manipulations would be apparent.…”

AI-guided screen identifies probucol-mediated mitophagy enhancement through modulation of lipid droplets

“…A recent report found that autophagy must be tuned to provide sufficient dynamic range to resolve differences in LC3 lipidation following manipulations such as drug treatments 35 . This can be accomplished by employing bafilomycin, an inhibitor of lysosome-autophagosome fusion at low doses so the effects of manipulations would be apparent.…”