Is LC3 a marker of autophagy?

LC3 (microtubule-associated protein 1 light chain 3) is a well-established marker of autophagy activity in cancer cells [5]. LC3 is a mammalian homolog of the yeast ATG8 protein, a ubiquitin-like protein that becomes lipidated and tightly associated with autophagosomal membranes [6].

Is LC3 degraded during autophagy?

Both LC3-II and p62 are degraded by autophagy; therefore, the amounts of LC3-II and p62 degraded by autophagy, but not their expression levels, provide an estimate of the autophagic activity.

How do you quantify autophagy?

Therefore, one of the best methods of quantifying autophagy is to measure the autophagic flux. This is how many autophagosomes form and then become degraded. To measure the flux, you need to detect the LC3-II turnover, or the difference in LC3-II levels in the presence versus the absence of lysosomal inhibitors.

What is LC3 Lipidation?

The final LC3 lipidation reaction is orchestrated by ATG7, ATG3, and a complex consisting of ATG12-ATG5 and ATG16L1 serving as E1, E2, and E3-like enzymes, respectively, to form LC3-II by conjugating the cytosolic LC3-I protein to PE.

How do you calculate autophagy flux?

Autophagosome flux can be quantified experimentally by completely blocking one of the steps in the pathway and measuring the initial rate of accumulation in the substrate for the inhibited step, such as, for example, the rate of autophagosome accumulation upon complete inhibition of the lysosomal fusion step.

How do you calculate autophagy?

What is BafA1?

Bafilomycin A1 (BafA1), a macrolide antibiotic isolated from Streptomyces species, is a specific vacuolar H+ ATPase (V-ATPase) inhibitor. Notably, due to its ability to specifically target V-ATPase and hence disrupt autophagic flux, BafA1 is frequently used to study autophagy and endosomal acidification.

What is autophagy flux?

The term “autophagic flux” is used to represent the dynamic process of autophagy. In detail, autophagic flux refers to the whole process of autophagy, including autophagosome formation, maturation, fusion with lysosomes, subsequent breakdown and the release of macromolecules back into the cytosol (Figure 1).