Measurement of Autophagy in Cells and Tissues - Protein Misfolding and Cellular Stress in Disease and Aging - page 188

Fig. 3. LC3-II is degraded by lysosomal proteases during autophagy. For nutrient-rich
conditions, cells were cultured in DMEM medium containing 10% FCS and non-essential
amino acids. Where indicated, cells were treated with the protease inhibitors, E64d
(10 m g/mL) and pepstatin A (10 m g/mL) (Inhibitors +) for 4 h, or with the solvent, dimeth-
ylsulfoxide, as a negative control (Inhibitors -). For starvation conditions, cells were incu-
bated for 4 h in Krebs-Ringer medium in the presence (+) or absence (−) of protease
inhibitors. The cells were lysed, total proteins (10 m g per lane) were separated by SDS-
PAGE, and endogenous LC3 and Atg7 in the lysates were recognized by immunoblotting
with anti-LC3 and anti-Atg7 antibodies, respectively. LC3-I, soluble form of LC3; LC3-II,
membrane-bound form of LC3. GAPDH was employed as a loading control. Note that
LC3-II accumulates under starvation conditions in the presence of the inhibitors.
defect in cells and tissues. p62 is a ubiquitin- and LC3-binding
protein that contributes to inclusion body formation. Autophagy-
deficient mouse brain and liver show accumulation of p62- and
ubiquitin-positive inclusions. Therefore, the increase in p62-positive
and ubiquitin-positive inclusions in tissues indicates the possibility
of an insufficiency in autophagy.
Various reagents are used for studying autophagy (Table 1 ).
mTor kinase negatively regulates autophagy via phosphorylation of
ULK1-Atg13-FIP200 complex ( 13-16 ). As rapamycin (sirolimus)
inhibits the activity of mTor kinase (mTORC1 complex) via bind-
ing to FKBP12, it induces autophagy. Torin1, another inhibitor of
mTor kinase, induces autophagy more strongly, as it directly inhib-
its mTor kinase (both mTORC1 and mTORC2 complexes) ( 17 ).
Lithium chloride (an inhibitor of IMPase), L-690 330 (another
IMPase inhibitor), and trehalose induce autophagy via an mTor-
independent pathway ( 18 ). Reagents that induce ER-stress (e.g.
brefeldin A and tunicamycin) also induce autophagy.