International Journal of Cell Biology LC3-II migrates more rapidly in SDS-PAGE compared to LC3-I, likely due to its higher hydrophobicity associated with the phosphatidylethanolamine group. However, LC3- II itself is degraded by autophagy, making interpretation of LC3 immunoblotting results problematic. Furthermore, the amount of LC3 at a certain time point does not indicate autophagic ﬂux. Monitoring of the natural autophagic substrate p62 (also called sequestosome 1) has been widely used to assess autophagic ﬂux. The degradation of GFP- LC3 was clearly less sensitive for inducers and activators of autophagy than GFP-p62 [93]. A reduction in the abundance of p62 due to its sequestration into autophagosomes, which results from the direct molecular interaction between LC3 and the LC3 interacting motifs (LIR) of p62, a 22-amino- acid acidic peptide motif, is interpreted as a sign of increased autophagy [94]; however, endogenous p62 is not produced at a constant level because some cellular stress conditions lead to induction of p62 at the transcriptional level, meaning that the level of p62 proteins is not solely determined by its turnover. Earlier autophagy studies relied on cell staining and ﬂuorescent microscopy. In particular, the overexpression of GFP-LC3, in which GFP (green ﬂuorescent protein) is expressed as a fusion protein at the amino terminus of LC3, was widely used to measure autophagy. These studies are limited, however, by several issues as follows: incorporation into protein that aggregates independent of autophagy, induction of autophagy by transfection proce- dures, sensitivity of GFP-LC3 to acid pH, and cessation of ﬂuorescence [95]. GFP is more resistant than LC3 in response to lysosomal degradation. When analyzing autophagic ﬂux based on GFP-LC3 degradation, the accumulation of free GFP and GFP-LC3-II in the absence and presence of a lyso- somal inhibitor should be compared. These may represent a serious problem in experiments testing the autophagic process. Many compounds to regulate autophagy are limited by the uncertainty as to whether they speciﬁcally target one step of the autophagy process. It is of interest to compare relative drug eﬀects obtained under diﬀerent settings, including conditions, time points, and concentrations. The compound 3-MA has been touted as an autophagy inhibitor but requires mM concentrations to inhibit class III PI3Ks involved in autophagy. 3-MA not only arrests autophagy but also prevents apoptosis by inhibiting the release of cytochrome c and cathepsin B. Rapamycin, commonly applied to induce autophagy, enhanced autophagy under full medium but had a surprising inhibitory eﬀect under nutrient deprivation conditions. Baﬁlomycin A1 is also commonly employed to suppress autophagic ﬂux due to its inhibition of the vacuolar type H+-ATPase (V-ATPase), blocking acidiﬁcation of lysosomes and endosomes. However, long-term treatment (>4 h) of cells with baﬁlomycin A1 also interferes with the traﬃcking of proteosomes, endosomes, and other cellular processes [96]. Therefore, a need exists for chemicals that target speciﬁc components of the autophagy machinery for use as research tools, to address questions about autophagy mechanisms and investigate the role of autophagy in cancers. 11 11. Concluding Remarks In this paper, the molecular players and mechanisms asso- ciated with autophagy have been described. Although autophagy is an important mechanism used by tumor cells to tolerate metabolic stress and is involved in nearly all stages of tumorigenesis and tumor progression, the issue of whether autophagy is aimed primarily at cell survival or at cell death is likely to remain a subject of debate for some time. As autophagy is a dynamic process that is diﬃcult to measure and quantify, new techniques for assessing autophagy need to be developed to ensure progress in this area of investigation. The multifaceted nature of autophagy and its diverse cross- talk with other biological processes must be carefully consid- ered when the autophagic system is targeted for anticancer beneﬁt. Acknowledgments The author apologizes to all colleagues whose work may not have been cited for space reasons. He is grateful to D. Mrozek for editing the manuscript. References [1] B. 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