Table 1.

Genetic and pharmacological regulation of autophagy.¹

Method		Comments
1.	3-methyladenine	A PtdIns3K inhibitor that effectively blocks an early stage of autophagy by inhibiting the class III PtdIns3K, but not a specific autophagy inhibitor. 3-MA also inhibits the class I PI3K and can thus, at suboptimal concentrations in long-term experiments, promote autophagy in some systems, as well as affect cell survival through AKT and other kinases. 3-MA does not inhibit BECN1-independent autophagy.
2.	10-NCP	10-(4′-N-diethylamino)butyl)-2-chlorophenoxazine; an AKT inhibitor that induces autophagy in neurons.¹²⁰⁰
3.	17-AAG	An inhibitor of the HSP90-CDC37 chaperone complex; induces autophagy in certain systems (e.g., neurons), but impairs starvation-induced autophagy and mitophagy in others by promoting the turnover of ULK1.⁴⁵⁸
4.	Akti-1/2	An allosteric inhibitor of AKT1 and AKT2 that promotes autophagy in B-cell lymphoma.¹⁴⁹⁵
5.	AR7	AR7 was developed as a highly potent and selective enhancer of CMA through antagonizing RARA/RARα; AR7 is the first small molecule developed to selectively stimulate CMA without affecting macroautophagy.¹⁴⁹⁶
6.	ARN5187	Lysosomotropic compound with a dual inhibitory activity against the circadian regulator NR1D2/REV-ERBβ and autophagy.¹⁴⁹⁷
7.	ATG4^C74A	An active site mutant of ATG4 that is defective for autophagy.¹⁴⁹⁸
8.	Bafilomycin A₁	A V-ATPase inhibitor that causes an increase in lysosomal/vacuolar pH, and, ultimately, blocks fusion of autophagosomes with the vacuole; the latter may result from inhibition of ATP2A/SERCA.²²⁶
9.	Betulinic acid	A pentacyclic triterpenoid that promotes paralell damage in mitochondrial and lysosomal compartments, and, ultimately, jeopardizes lysosomal degradative capacity.²³⁵
10.	Calcium	An autophagy activator that can be released from ER or lysosomal stores under stress conditions; however, calcium can also inhibit autophagy.^216,1245
11.	Chloroquine, NH₄Cl	Lysosomotropic compounds that elevate/neutralize the lysosomal/vacuolar pH.¹⁶³
12.	DFMO	α-difluoromethylornithine, an irreversible inhibitor of ODC1 (ornithine decarboxylase 1) that blocks spermidine synthesis and ATG gene expression.¹⁴⁹⁹
13.	E-64d	A membrane-permeable cysteine protease inhibitor that can block the activity of a subset of lysosomal hydrolases; should be used in combination with pepstatin A to inhibit lysosomal protein degradation.
14.	ESC8	A cationic estradiol derivative that induces autophagy and apoptosis simultaneously by downregulating the MTOR kinase pathway in breast cancer cells.
15.	Everolimus	An inhibitor of MTORC1 that induces both autophagy and apoptosis in B-cell lymphoma primary cultures.¹⁴⁹⁵
16.	Fumonisin B1	An inhibitor of ceramide synthesis that interferes with macroautophagy.
17.	Gene deletion	This method provides the most direct evidence for the role of an autophagic component; however, more than one gene involved in autophagy should be targeted to avoid indirect effects.
18.	HMOX1 induction	Mitophagy and the formation of iron-containing cytoplasmic inclusions and corpora amylacea are accelerated in HMOX1-transfected rat astroglia and astrocytes of GFAP-HMOX1 transgenic mice. Heme derived ferrous iron and carbon monoxide, products of the HMOX1 reaction, promote macroautophagy in these cells.^1500-1502
19.	Knockdown	This method (including miRNA, RNAi, shRNA and siRNA) can be used to inhibit gene expression and provides relatively direct evidence for the role of an autophagic component. However, the efficiency of knockdown varies, as does the stability of the targeted protein. In addition, more than one gene involved in autophagy should be targeted to avoid misinterpreting indirect effects.
20.	KU-0063794	An MTOR inhibitor that binds the catalytic site and activates autophagy.^341,1503
21.	Leupeptin	An inhibitor of cysteine, serine and threonine proteases that can be used in combination with pepstatin A and/or E-64d to block lysosomal protein degradation. Leupeptin is not membrane permeable, so its effect on cathepsins may depend on endocytic activity.
22.	microRNA	Can be used to reduce the levels of target mRNA(s) or block translation.
23.	MLN4924	A small molecule inhibitor of NAE (NEDD8 activating enzyme);¹⁵⁰⁴ induces autophagy by blockage of MTOR signals via DEPTOR and the HIF1A-DDIT4/REDD1-TSC1/2 axis as a result of inactivation of CUL/cullin-RING ligases.^1505-1507
24.	NAADP-AM	Activates the lysosomal TPCN/two-pore channel and induces autophagy.¹²²⁵
25.	NED-19	Inhibits the lysosomal TPCN and NAADP-induced autophagy.¹²²⁵
26.	NVP-BEZ235	A dual inhibitor of PIK3CA/p110 and the MTOR catalytic site that activates autophagy.^1508,1509
27.	Pathogen-derived	Virally-encoded autophagy inhibitors including HSV-1 ICP34.5, Kaposi sarcoma-associated herpesvirus vBCL2, γ-herpesvirus 68 M11, ASFV vBCL2, HIV-1 Nef and influenza A virus M2.^{566,892,896,897,902}
28.	Pepstatin A	An aspartyl protease inhibitor that can be used to partially block lysosomal degradation; should be used in combination with other inhibitors such as E-64d. Pepstatin A is not membrane permeable.
29.	Protease inhibitors	These chemicals inhibit the degradation of autophagic substrates within the lysosome/vacuole lumen. A combination of inhibitors (e.g., leupeptin, pepstatin A and E-64d) is needed for complete blockage of degradation.
30.	PMI	p62 (SQSTM1)-mediated mitophagy inducer is a pharmacological activator of autophagic selection of mitochondria that operates without collapsing the mitochondrial membrane potential (ΔΨ_m) and hence by exploiting the autophagic component of the process.⁷¹³
31.	Rapamycin	Binds to FKBP1A/FKBP12 and inhibits MTORC1; the complex binds to the FRB domain of MTOR and limits its interaction with RPTOR, thus inducing autophagy, but only providing partial MTORC1 inhibition. Rapamycin also inhibits yeast TOR.
32.	Resveratrol	A natural polyphenol that affects many proteins¹⁵¹⁰ and induces autophagy via activation of AMPK.^1511,1512
33.	RNAi	Can be used to inhibit gene expression.
34.	RSVAs	Synthetic small-molecule analogs of resveratrol that potently activate AMPK and induce autophagy.¹⁵¹³
35.	Saikosaponin-d	A natural small-molecule inhibitor of ATP2A/SERCA that induces autophagy and autophagy-dependent cell death in apoptosis-resistant cells.¹⁵¹⁴
36.	Tat-Beclin 1	A cell penetrating peptide that potently induces macroautophagy.^1080,1226
37.	Thapsigargin	An inhibitor of ATP2A/SERCA that inhibits autophagic sequestration through the depletion of intracellular Ca²⁺ stores;^216,1515 however, thapsigargin may also block fusion of autophagosomes with endosomes by interfering with recruitment of RAB7, resulting in autophagosome accumulation.¹⁵¹⁶
38	TMS	Trans-3,5,4-trimethoxystilbene upregulates the expression of TRPC4, resulting in MTOR inhibition.¹⁵¹⁷
39.	Torin1	A catalytic MTOR inhibitor that induces autophagy and provides more complete inhibition than rapamycin (it inhibits all forms of MTOR).¹¹⁹³
40.	Trehalose	An inducer of autophagy that may be relevant for the treatment of different neurodegenerative diseases.^{1241,1518,1519}
41.	Tunicamycin	A glycosylation inhibitor that induces autophagy due to ER stress.¹⁵²⁰
42.	Vacuolin-1	A RAB5A activator that reversibly blocks autophagosome-lysosome fusion.¹⁵²¹
43.	Vinblastine	A depolymerizer of both normal and acetylated microtubules that interferes with autophagosome-lysosome fusion.²²⁷
44.	Wortmannin	An inhibitor of PI3K and PtdIns3K that blocks autophagy, but not a specific inhibitor (see 3-MA above).

This table is not meant to be complete, as there are many compounds and genetic methods that regulate autophagy, and new ones are being discovered routinely.