Guidelines for the use and interpretation of assays for monitoring autophagy

Abstract

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

MicroRNA‐21 protects against cardiac hypoxia/reoxygenation injury by inhibiting excessive autophagy in H9c2 cells via the Akt/mTOR pathway

“…As shown in Figure 2B, the number of autophagic vacuoles and autophagosomes in the H/R group was increased 2.4‐fold compared to the control group ( P < 0.001), and the miR‐21 precursor decreased this increase to approximately 1.5‐fold ( P < 0.001) in the (miR‐21+ H/R) group. Figure 2C and E showed that miR‐21 up‐regulation resulted in a decrease in the ratio of LC3‐II/LC3‐I compared to the H/R group (51.2 ± 13.3% versus 197.3 ± 37.0%, P = 0.003), which is a reliable indicator of autophagy 21. A decrease in the levels of the ubiquitin‐binding protein p62 occurs when autophagy is activated because p62 is an autophagy substrate 21.…”

AMPK activation protects cells from oxidative stress‐induced senescence via autophagic flux restoration and intracellular NAD + elevation

“…3C). To examine the status of autophagic flux, a NIH3T3 cell population stably expressing a tandem RFP‐GFP‐LC3 fusion protein was established and employed to visualize and distinguish GFP+RFP+ (yellow) and GFP‐GFP+ (red) LC3 puncta (Klionsky et al ., 2012). As shown in Fig.…”

“…Despite the fact that the number of autophagosomes increased in senescent cells, our result strongly supports the notion that autolysosomal degradation and autophagic flux were attenuated in these cells. To explore the status of autophagic flux, we applied several reliable assays (Mizushima et al ., 2010; Klionsky et al ., 2012), such as evaluating the Cathepsin B protein level, measuring the acid phosphatase activity, and comparing the influence of a lysosome inhibitor on the p62 accumulation. We also assessed the inhibition of GFP‐LC3 fluorescence using a GFP‐RFP‐LC3‐expressing cell, and obtained consistent data with previous report (Burkewitz K et al ., 2014), indicating that AMPK activation can improve the autophagic activity in cells with H 2 O 2 ‐induced senescence.…”

“…Autophagy is a homeostatic cellular recycling mechanism responsible for degrading injured or dysfunctional cellular organelles and proteins in all living cells (Mizushima et al ., 2010). The dynamic process of autophagy is usually surveyed by determining the autophagic flux (Klionsky et al ., 2012). Growing evidence has indicated that the rate of autophagosome formation/maturation and the efficiency of autophagosome/lysosome fusion decline with age (Mijaljica et al ., 2010).…”

“…Growing evidence has indicated that the rate of autophagosome formation/maturation and the efficiency of autophagosome/lysosome fusion decline with age (Mijaljica et al ., 2010). The methods used to monitor autophagic flux include evaluations of the degradation of p62 protein and assessment of the activity of autolysosomal hydrolases (Klionsky et al ., 2012), as well as examining the quenching of GFP‐tagged LC3 protein (Kimura et al ., 2007). …”