10 International Journal of Cell Biology of LC3-II-associated autophagosomes. Strong inhibition of autophagic vacuole accumulation was found in nocodazole- arrested pseudoprometaphase cells. The loss of microtubules induced by nocodazole treatment results in the disconnection of Golgi stacks and dispersal into 70–100 smaller fragments [84]. relevant autophagy inhibitors that block lysosyme acidiﬁ- cation and degradation of autophagosomes [89]. Chloro- quine, through its lysosomotropic properties, as well as its autophagy inhibition ability, may be a promising agent to be used in combination with chemotherapeutic agents to improve clinical results. 9.2.7. Heat Shock Proteins. Heat shock protein 90 (Hsp90) forms a protein complex to maintain the stability of its client proteins. Disruption of this protein complex with speciﬁc Hsp90 inhibitors leads to proteolytic degradation of the client proteins, usually through the ubiquitin- proteasome pathway. Hsp90 forms a complex with Beclin 1 through an evolutionarily conserved domain to maintain the stability of Beclin 1. Geldanamycin, an Hsp90 inhibitor, eﬀectively promoted proteasomal degradation of Beclin 1 in a concentration-dependent and time-dependent manner [85]. A small molecule called 2-phenylethynesulfonamide (synonym, also called piﬁthrin-μ, PES) interacts selectively with Hsp70 and leads to a disruption of the association between Hsp70 and several of its cochaperones and substrate proteins. PES was identiﬁed as a novel Hsp70 inhibitor. PES- mediated inhibition of Hsp70 family proteins in tumor cells results in impairment of the autophagy-lysosome system. PES impaired the mitochondrial localization of p53 [86]. The p14/p19ARF tumor suppressor gene is frequently mutated in human cancer. ARF (alternative reading frame) has been shown to localize to mitochondria and to induce autophagy. Treatment of cells with PES blocks the traﬃcking of ARF to mitochondria. 9.2.8. Lysosomal Function. Baﬁlomycin A1 is in the pleco- macrolide subclass of macrolide antibiotics, and a highly speciﬁc inhibitor of V-ATPase, whereas other types (F- type, P-type, Ca2+, and K+) of ATPases are not aﬀected by this antibiotic. The anticancer eﬀect of baﬁlomycin A1 is well known and is attributed mainly to the inhibition of autophagy by preventing the fusion of autophagosomes with dysfunctional lysosomes, consequently triggering apop- tosis [87]. Lucanthone (miracil D) has been extensively used as an antischistome agent. The drug also blocks topoisomerase II activity and has been reported to inhibit AP (apurinic/apyrimidinic) endonuclease, an important enzyme in DNA base excision repair. Lucanthone inhibits autophagy, caused by induction of lysosomal membrane permeabilization. Lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat [88]. Lucanthone is currently being investigated as a sensitizer to chemotherapy and radiation due to its ability to interfere with DNA repair. Chloroquine (avloclor) is a well-known 4-aminoquinoline class drug that is widely used for prophylaxis treatment against malaria. Pharmacologically, chloroquine is a weak base and is trapped in acidic organelles like lysosomes, resulting in increased vacuolar pH6.0. Chloroquine and its analog hydroxychloroquine (plaquenil) are the only clinically 9.2.9. Small Regulatory RNAs. Small regulatory RNAs have become a speciﬁc and powerful tool to turn oﬀ the expression of target genes. Their actions include the suppression of overexpressed oncogenes, retarding cell division by inter- fering with cyclins and related genes or enhancing apop- tosis by inhibiting anti-apoptotic genes. Paclitaxel, which stabilizes microtubules and causes apoptosis, oﬀers both symptomatic and survival beneﬁts for lung adenocarci- noma. Both autophagy and apoptosis are induced in cancer cells during the course of paclitaxel treatment. Paclitaxel treatment could lead to the formation of acidic vesicular organelles and the induction of Atg5, Beclin 1, and LC3 expression. Paclitaxel-mediated apoptotic cell death was further potentiated by pretreatment with Beclin 1 siRNA [90]. Lysosomal-associated membrane protein 2 (Lamp-2) is a ubiquitous lysosomal membrane protein that is highly expressed in normal human pancreatic tissue and is required for the proper fusion of lysosomes with autophagosomes in the late stage of the autophagic process. Silencing with a speciﬁc Lamp-2 siRNA abolished immunoﬂuorescence particles for Lamp-2, conﬁrming depletion of Lamp-2 protein. LC3-positive immunoﬂuorescence puncta signiﬁ- cantly increased in Lamp-2-silenced cells, suggesting that Lamp-2 depletion correlated with the accumulation of autophagosomes and a relative paucity of autolysosomes in pancreatic acinar cells. Lamp-2 depletion causes a “traﬃc jam” that culminates in the accumulation of autophago- somes [91]. MicroRNAs (miRNAs) are a class of endoge- nous, 22–24 nucleotide RNA molecules with the ability to induce mRNA degradation, translational repression, or both, via pairing with partially complementary sites in the 3 UTR of the targeted genes. miRNAs can control the expression of autophagic genes, thereby modulating autophagic activity. Indeed, inhibition of the expression by beclin 1 miR-30a leads to suppression of autophagic activity [92]. 10. Technical Errors in Autophagy Study One of the technical problems in the study of autophagy is the lack of convenient and reliable methods to detect it. Uncertainty in the conﬁrmation of the fully functional autophagic process could be one of the causes of confusion related to the role of autophagy in cell death. These types of confusion and diﬃculties are more pronounced in animal experiments that are critical in the study of cancers. LC3 is now widely used to monitor autophagy. One approach is to detect LC3 conversion (LC3-I to LC3- II) by immunoblot analysis because the amount of LC3- II is clearly correlated with the number of autophago- somes. Despite a higher molecular weight than LC3-I,