Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)¹

Autophagy in Hepatic Steatosis: A Structured Review

“…Both proteins are widely used as monitors of autophagy activation/flux in the literature; LC3-II is the molecular signature of autophagosome formation, and binds to p62, favoring cargo capture for degradation (Pankiv et al, 2007). We highlight that comparisons must be interpreted with caution, as thoroughly discussed by Klionsky et al (2021).…”

Yersiniabactin Siderophore of Crohn’s Disease-Associated Adherent-Invasive Escherichia coli Is Involved in Autophagy Activation in Host Cells

“…As we have previously shown a crucial role for autophagy in the control of AIEC intracellular replication [21,[24][25][26][27], we next investigated whether autophagy is implicated in the increase of LF82 intracellular replication associated with the loss of yersiniabactin. Autophagy was assessed by Western blot analysis by evaluating the shift of LC3-I (the cytosolic form) toward LC3-II (the autophagosomal form and a marker of autophagy induction) and the level of p62, an autophagy receptor which is degraded by a functional autophagy [42]. As expected, autophagy was induced in T84 cells upon infection with the wild-type LF82 strain, as evidenced by increased LC3-II level compared with uninfected cells (Figure 3b).…”

“…These observations suggested a link between the presence of a functional yersiniabactin siderophore and the activation of autophagy in infected cells. The increase in LC3-II levels could result from the activation of autophagy or a blockage of autophagy flux, leading to the accumulation of undegraded LC3-II [42]. Thus, to examine whether the presence of yersiniabactin siderophore led to autophagy activation or autophagy flux blockage, we treated the cells with the inhibitor of autophagy flux bafilomycin A1 before infection.…”

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

“…To monitor autophagy, we used indirect immunofluorescence microscopy to score the formation of punctate intracellular vacuoles stained for LC3B. Moreover, to better appreciate autophagosomal formation, we also used bafilomycin A1 (Baf A1), an inhibitor of lysosomal acidification, to prevent lysosomal degradation of autophagosome-associated LC3B [34]. Baf A1 allows, in fact, the detection of each autophagosome formed in the time-lapse between addition of the drug to cells and harvesting [34].…”

“…Moreover, to better appreciate autophagosomal formation, we also used bafilomycin A1 (Baf A1), an inhibitor of lysosomal acidification, to prevent lysosomal degradation of autophagosome-associated LC3B [34]. Baf A1 allows, in fact, the detection of each autophagosome formed in the time-lapse between addition of the drug to cells and harvesting [34]. Importantly, to avoid potentially confounding effects of the endogenous TLR3 receptor in transfected cells, we used TLR3 knock-out HEK cells.…”