Methodological considerations for assessing autophagy modulators: a study with calcium phosphate precipitates

Autophagy has been implicated in various physiological and disease conditions in recent years. A number of small molecule modulators have been identified, both as tools and as potential therapeutics. Despite extensive characterization of autophagy in yeast, mammalian autophagy pathways are not fully understood. Recently, calcium phosphate precipitates (CPP), which are used to transfect DNA into cells, were reported to induce autophagy, when assayed up to 6 h after treatment. Because of the widespread use of this reagent, we attempted to confirm these findings. Consistent with the previous study, we showed that CPP induces autophagosome synthesis at early time points, such as 4 h and 6 h. However, at 24 h after treatment, we were surprised to see that autophagy flux was reduced, due to impaired autophagosome-lysosome fusion. At this time point, there was an accumulation of autophagy substrates and the formation of abnormally large autophagosomes. Thus, one may need to consider assaying autophagy modulators at different time points with a range of assays in order to understand their effects. Finally, the complex consequences of CPP on autophagy suggest that it is best avoided as a transfection reagent in studies aiming to analyze autophagy itself, or processes that are modulated by autophagy, like apoptosis.