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

The mechanism of apoptosis has been extensively characterized over the past decade, but little is known about alternative forms of regulated cell death. Although stimulation of the Fas/TNFR receptor family triggers a canonical 'extrinsic' apoptosis pathway, we demonstrated that in the absence of intracellular apoptotic signaling it is capable of activating a common nonapoptotic death pathway, which we term necroptosis. We showed that necroptosis is characterized by necrotic cell death morphology and activation of autophagy. We identified a specific and potent small-molecule inhibitor of necroptosis, necrostatin-1, which blocks a critical step in necroptosis. We demonstrated that necroptosis contributes to delayed mouse ischemic brain injury in vivo through a mechanism distinct from that of apoptosis and offers a new therapeutic target for stroke with an extended window for neuroprotection. Our study identifies a previously undescribed basic cell-death pathway with potentially broad relevance to human pathologies.

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Caspases are a family of cysteine proteases that play important roles in regulating apoptosis. A decade of research has generated a wealth of information on the signal transduction pathways mediated by caspases, the distinct functions of individual caspases and the mechanisms by which caspases mediate apoptosis and a variety of physiological and pathological processes.

Stimulation of death receptors by agonists such as FasL and TNFα activates apoptotic cell death in apoptotic competent conditions or a type of necrotic cell death dependent on RIP1 kinase, termed necroptosis, in apoptotic deficient conditions. In a genome-wide siRNA screen for regulators of necroptosis, we identify a set of 432 genes that regulate necroptosis, a subset of 32 genes that act downstream and/or as regulators of RIP1 kinase, 32 genes required for death receptor mediated apoptosis, and 7 genes involved in both necroptosis and apoptosis. We show that the expression of subsets of the 432 genes are enriched in the immune and nervous systems, and cellular sensitivity to necroptosis is regulated by an extensive signaling network mediating innate immunity. Interestingly, Bmf, a BH3-only Bcl-2 family member, is required for death receptor-induced necroptosis. Our study defines a cellular signaling network that regulates necroptosis and the molecular bifurcation that controls apoptosis and necroptosis.

Assembly of the eIF4E/eIF4G complex has a central role in the regulation of gene expression at the level of translation initiation. This complex is regulated by the 4E-BPs, which compete with eIF4G for binding to eIF4E and which have tumor-suppressor activity. To pharmacologically mimic 4E-BP function we developed a high-throughput screening assay for identifying small-molecule inhibitors of the eIF4E/eIF4G interaction. The most potent compound identified, 4EGI-1, binds eIF4E, disrupts eIF4E/eIF4G association, and inhibits cap-dependent translation but not initiation factor-independent translation. While 4EGI-1 displaces eIF4G from eIF4E, it effectively enhances 4E-BP1 association both in vitro and in cells. 4EGI-1 inhibits cellular expression of oncogenic proteins encoded by weak mRNAs, exhibits activity against multiple cancer cell lines, and appears to have a preferential effect on transformed versus nontransformed cells. The identification of this compound provides a new tool for studying translational control and establishes a possible new strategy for cancer therapy.

Cell death has historically been subdivided into regulated and unregulated mechanisms. Apoptosis, a form of regulated cell death, reflects a cell's decision to die in response to cues and is executed by intrinsic cellular machinery. Unregulated cell death (often called necrosis) is caused by overwhelming stress that is incompatible with cell survival. Emerging evidence, however, suggests that these two processes do not adequately explain the various cell death mechanisms. Recent data point to the existence of multiple non-apoptotic, regulated cell death mechanisms, some of which overlap or are mutually exclusive with apoptosis. Here we examine how and why these different cell death programmes have evolved, with an eye towards new cytoprotective therapeutic opportunities.

Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.