In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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A branched-chain polyunsaturated fatty acid, geranylgeranoic acid (GGA; C20:4), which is an endogenous metabolite derived from the mevalonate pathway in mammals, has been reported to induce cell death in human hepatoma cells. We have previously shown that the lipid-induced unfolded protein response (UPR) is an upstream cellular process for an incomplete autophagic response that might be involved in GGA-induced cell death. Here, we found that Toll-like receptor 4 (TLR4)-mediated pyroptosis in HuH-7 cells occurred by GGA treatment. The TLR4-specific inhibitor VIPER prevented both GGA-induced cell death and UPR. Knockdown of the TLR4 gene attenuated GGA-induced cell death significantly. Upon GGA-induced UPR, caspase (CASP) 4 (CASP4) was activated immediately and gasdermin D (GSDMD) was translocated concomitantly to the plasma membrane after production of the N-terminal fragment of GSDMD. Then, cellular CASP1 activation occurred following a second gradual up-regulation of the intracellular Ca2+ concentration, suggesting that GGA activated the inflammasome. Indeed, the mRNA levels of NOD-like receptor family pyrin domain containing 3 (NLRP3) and interleukin-1 β (IL1B) genes were up-regulated dramatically with translocation of cytoplasmic nuclear factor-κB (NF-κB) to nuclei after GGA treatment, indicating that GGA induced priming of the NLRP3 inflammasome through NF-κB activation. GGA-induced up-regulation of CASP1 activity was blocked by either oleic acid, VIPER, MCC950 (a selective inhibitor of the NLRP3 inflammasome), or CASP4-specific inhibitor peptide cotreatment. Pyroptotic cell death was also confirmed morphologically by bleb formation in time-series live cell imaging of GGA-treated cells. Taken together, the present results strongly indicate that GGA causes pyroptotic cell death in human hepatoma-derived HuH-7 via TLR4 signalling.

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All-trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid (designated "acyclic retinoid") induced upregulation of the albumin gene expression at its transcriptional level, whereas all-trans-retinoic acid (RA) induced downregulation of the expression in both PLC/PRF/5 and HuH7 human hepatoma cell lines. These up- and down regulations of the albumin gene expression coordinated with high and low levels of mRNA for hepatocyte nuclear factor-1 (HNF-1), which is one of the most potent transcription factors for the albumin gene, implying that retinoids may regulate albumin gene expression through HNF-1 expression in opposite ways. The PLC/PRF/5 and HuH7 hepatoma cell lines expressed retinoid X receptor-alpha (RXR alpha) mRNA, whose expression was constitutive. Acyclic retinoid and all-trans-RA both induced upregulation of retinoic acid receptor-beta (RAR beta), and both suppressed cell proliferation-related phenotypic expressions by the alpha-fetoprotein gene and the c-myc oncogene. 9-cis-RA, whose receptor is known to be RXR alpha, also induced upregulation of albumin and HNF-1 expression. These results suggest that acyclic retinoid may act through both RXR alpha and RAR beta, whereas all-trans-RA conveys only RAR beta-mediated functions, at least in these two hepatoma cell lines.

GGA (geranylgeranoic acid) is a natural polyprenoic acid, derivatives of which has been shown to prevent second primary hepatoma. GGA induces mitochondria-mediated PCD (programmed cell death), which may be relevant to cancer prevention. To gain further insights into GGA-induced PCD, autophagy processes were examined in human hepatoma-derived HuH-7 cells. Treatment of HuH-7/GFP (green fluorescent protein)-LC3 cells with GGA induced green fluorescent puncta in the cytoplasm within 30 min and their massive accumulation at 24 h. After 15 min of GGA treatment, a burst of mitochondrial superoxide production occurred and LC3β-I was appreciably converted into LC3β-II. GGA-induced early stages of autophagy were unequivocally confirmed by electron-microscopic observation of early/initial autophagic vacuoles. On the other hand, LC3β-II as well as p62/SQSTM1 (sequestosome 1) continuously accumulated and co-localized in the cytoplasmic puncta after GGA treatment. Furthermore, GGA treatment of HuH-7/mRFP (monomeric red fluorescent protein)-GFP-LC3 cells showed yellow fluorescent puncta, whereas glucose deprivation of the cells gave red fluorescent puncta. These results strongly suggest that GGA induces the initial phase of autophagy, but blocks the maturation process of autolysosomes or late stages of autophagy, insomuch that GGA provides substantial accumulation of autophagosomes under serum-starvation conditions in human hepatoma cells.