Measurement of Autophagy in Cells and Tissues - Protein Misfolding and Cellular Stress in Disease and Aging - page 190

Table 1
Autophagy inducers, inhibitors, and media for starvation

Reagents that induce autophagy Stock solution Final conc.

Rapamycin 1 mM in DMSO or ethanol, -20°C 0.5-1 mM

Torin1 1 mM in DMSO, -20°C 1 mM

Lithium chloride 1 M in H 2 O, 4°C 10 mM

L-690,330 1 M in H 2 O, 4°C 100 mM

Trehalose powder 100 mM

Reagents that inhibit autophagy Stock solution Final conc.

3-MA powder 5-10 mM

E64d and pepstatin A 10 mg/ml each in DMSO, -20°C 10 mg/ml

Bafilomycin A1 0.1 mM in DMSO, -20°C 0.1 mM
E64d is membrane-permeable, while E64, E64c, and leupeptin are membrane-impermeable (see Note 1). When
leupeptin is used instead of E64d and pepstatin A as cathepsin inhibitors, care should be taken to account for its
membrane impermeability

the activity of the delivery process of LC3-II into lysosomes.
Thus, these drugs can be used as tools for measurement of
autophagy flux. Leupeptin, a membrane-impermeable inhibitor
of cathepsin B, is used for investigation of autophagy in the liver
( 19 ), but only rarely in other tissues, as leupeptin accumulates
effectively in the liver probably via endocytosis. Therefore, it is
possible that leupeptin inhibits cathepsin B in some cells and tissues
via the endocytic pathway. As leupeptin is a membrane-imperme-
able reagent, it should be confirmed whether this drug actually
inhibits cathepsin B in cells and tissues before studying
autophagy. Bafilomycin A1 is an inhibitor of V-ATPase that is
essential for acidification of lysosomes. Therefore, this drug is
also used for inhibition of autophagy and estimation of
autophagic flux of LC3-II. As V-ATPase contributes to the
acidification of the other organelles, including the Golgi and
endosomes, bafilomycin A1 potentially shows multiple off-target
effects ( 20 ). Rab7, one of the small GTPases, mediates the fusion
of autophagosomes with lysosomes ( 21, 22 ). A dominant negative
mutant of rab7, rab7 T22N , inhibits this fusion step. Overexpression
of this rab7 mutant results in augmented accumulation of autopha-
gosomes under starvation conditions. However, rab7 is not specific
for autophagosome-lysosome fusion, as it also plays an indispens-
able role in endosome-lysosome fusion.