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.

For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.

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Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

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The MLL recombinome of acute leukemias in 2017

Modulating ROS to overcome multidrug resistance in cancer.

The vacuolar ATPases (V-ATPases) are a family of proton pumps that couple ATP hydrolysis to proton transport into intracellular compartments and across the plasma membrane. They function in a wide ...

/pdf/the-function-of-v-atpases-in-cancer-39a1jiqnz0.pdf

The Function of V-ATPases in Cancer

COVID-19, a global pandemic, has caused over 750,000 deaths worldwide as of August 2020. A vaccine or remedy for SARS-CoV-2, the virus responsible for COVID-19, is necessary to slow down the spread and lethality of COVID-19. However, there is currently no effective treatment available against SARS-CoV-2. In this report, we demonstrated that EGCG and theaflavin, the main active ingredients of green tea and black tea, respectively, are potentially effective to inhibit SARS-CoV-2 activity. Coronaviruses require the 3CL-protease for the cleavage of its polyprotein to make individual proteins functional. EGCG and theaflavin showed inhibitory activity against the SARS-CoV-2 3CL-protease in a dose-dependent manner, and the half inhibitory concentration (IC50) was 7.58 μg/ml for EGCG and 8.44 μg/ml for theaflavin. In addition, we did not observe any cytotoxicity for either EGCG or theaflavin at the concentrations tested up to 40 μg/ml in HEK293T cells. These results suggest that upon further study, EGCG and theaflavin can be potentially useful to treat COVID-19.

/pdf/tea-polyphenols-egcg-and-theaflavin-inhibit-the-activity-of-4gyjdd8w83.pdf

Tea Polyphenols EGCG and Theaflavin Inhibit the Activity of SARS-CoV-2 3CL-Protease In Vitro .

AMP-activated protein kinase (AMPK) regulates autophagy initiation when intracellular ATP level decreases. However, the role of AMPK during autophagosome maturation is not fully understood. Here, we report that AMPK contributes to efficient autophagosome maturation and lysosomal fusion. Using CRISPR-Cas9 gene editing, we generated AMPK α1 knockout HEK293T cell lines, in which starvation-induced autophagy is impaired. Compound C, an AMPK-independent autophagy inducer, and trehalose, an mTOR-independent autophagy inducer were used to examine the role of AMPK in autophagosome maturation and lysosomal fusion. While the treatment of control cells with either compound C or trehalose induces activation of autophagosomes as well as autolysosomes, the treatment of AMPK α1 knockout cells with compound C or trehalose induces mainly activation of autophagosomes, but not autolysosomes. We demonstrate that this effect is due to interference with the fusion of autophagosomes with lysosomes in AMPK α1 knockout cells. The transient expression of AMPK α1 can rescue autophagosome maturation. These results indicate that AMPK α1 is required for efficient autophagosome maturation and lysosomal fusion.

/pdf/ampk-contributes-to-autophagosome-maturation-and-lysosomal-15a1w6bnwv.pdf

AMPK contributes to autophagosome maturation and lysosomal fusion

EGCG, a green tea polyphenol, inhibits human coronavirus replication in vitro.

The PKA-CREB signaling pathway is involved in many cellular processes including autophagy. Recent studies demonstrated that PKA-CREB inhibits autophagy in yeast; however, the role of PKA-CREB signaling in mammalian cell autophagy has not been fully characterized. Here, we report that the integral membrane protein ITM2A expression is positively regulated by PKA-CREB signaling and ITM2A expression interferes with autophagic flux by interacting with vacuolar ATPase (v-ATPase). The ITM2A promoter contains a CRE element, and mutation at the CRE consensus site decreases the promoter activity. Forskolin treatment and PKA expression activate the ITM2A promoter confirming that ITM2A expression is dependent on the PKA-CREB pathway. ITM2A expression results in the accumulation of autophagosomes and interferes with autolysosome formation by blocking autophagic flux. We demonstrated that ITM2A physically interacts with v-ATPase and inhibits lysosomal function. These results support the notion that PKA-CREB signaling pathway regulates ITM2A expression, which negatively regulates autophagic flux by interfering with the function of v-ATPase.

The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase

Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.

/pdf/crispr-cas9-mediated-nox4-knockout-inhibits-cell-2nx7ffy62x.pdf

Rackhyun Park

Papers

Tea Polyphenols EGCG and Theaflavin Inhibit the Activity of SARS-CoV-2 3CL-Protease In Vitro .

AMPK contributes to autophagosome maturation and lysosomal fusion

EGCG, a green tea polyphenol, inhibits human coronavirus replication in vitro.

The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase

CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells