Real-time visualization and quantitation of cell death and cell cycle progression in 2D and 3D cultures utilizing genetically encoded probes.

Cancer cells grown as 3D-structures are better models for mimicking in vivo conditions than the 2D-culture systems employable in drug discovery applications. Cell cycle and cell death are important determinants for preclinical drug screening and tumor growth studies in laboratory conditions. Though several 3D-models and live-cell compatible approaches are available, a method for simultaneous real-time detection of cell cycle and cell death is required. Here we demonstrate a high-throughput adaptable method using genetically encoded fluorescent probes for the real-time quantitative detection of cell death and cell cycle. The cell-cycle indicator cdt1-Kusabira orange (KO) is stably integrated into cancer cells and further transfected with the Fluorescence Resonance Energy Transfer-based ECFP-DEVD-EYFP caspase activation sensor. The nuclear cdt1-KO expression serves as the readout for cell-cycle, and caspase activation is visualized by ECFP/EYFP ratiometric imaging. The image-based platform allowed imaging of growing spheres for prolonged periods in 3D-culture with excellent single-cell resolution through confocal microscopy. High-throughput screening (HTS) adaptation was achieved by targeting the caspase-sensor at the nucleus, which enabled the quantitation of cell death in 3D-models. The HTS using limited compound libraries, identified two lead compounds that induced caspase-activation both in 2D and 3D-cultures. This is the first report of an approach for noninvasive stain-free quantitative imaging of cell death and cell cycle with potential drug discovery applications.

A high-throughput image-based screen for the identification of Bax/Bak-independent caspase activators against drug-resistant cancer cells.

Despite the use of new generation target specific drugs or combination treatments, drug-resistance caused by defective apoptosis signaling remains a major challenge in cancer treatment. A common apoptotic defect in drug-resistant tumor is the failure of cancer cells to undergo Bax/Bak-dependent mitochondrial permeabilization due to impaired signaling of Bcl-2 family proteins. Therefore, Bax and Bak-independent caspase-activating compounds appear to be effective in killing such tumor cells. An image-based cellular platform of caspase sensors in Bax and Bak deficient background allowed us to identify several potential Bax/Bak-independent caspase-activating compounds from a limited high-throughput compound screening. FRET-based caspase sensor probe targeted at the nucleus enabled accurate and automated segmentation, yielding a Z-value of 0.72. Some of the positive hits showed promising activity against drug-resistant human cancer cells expressing high levels of Bcl-2 or Bcl-xL. Using this approach, we describe thiolutin, CD437 and TPEN as the most potentially valuable drug candidates for addressing drug-resistance caused by aberrant expression of Bcl-2 family proteins in tumor cells. The screen also enables the quantification of multiparameter apoptotic events along with caspase activation in HTS manner in live mode, allowing characterization of non-classical apoptosis signaling.

Genetically encoded caspase sensor and RFP-LC3 for temporal analysis of apoptosis-autophagy.

The balance between pro-death and pro-survival signaling determines the fate of cells under a variety of pathological and physiological conditions. The pro-cell death signaling, apoptosis, and survival singling, autophagy work in an integrated manner for maintaining cell integrity. Their altered balance drives pathological conditions such as cancer, inflammatory disorders, and neurodegenerative diseases. Dissecting complex crosstalk between autophagy and apoptosis requires simultaneous detection of both events at a single cell level with good temporal resolution in real-time. Here, we have used two distinct fluorescent-based probes of caspase activation and autophagy for generating such sensor cells. Cells stably expressing RFP-LC3 as an autophagy marker were further stably expressed with a FRET-based probe for caspase activation with a nuclear localization signal. The functional validation and live-cell imaging of the sensor cells using selected treatments revealed that stress that induces rapid cell death often fails to induce autophagy signaling, and slow cell death induction triggers simultaneous autophagy signaling with caspase activation. The real-time imaging revealed the time-dependent shift of cells towards caspase activation while autophagy is inhibited confirming basal autophagy confers survival against apoptosis under stress conditions. Confocal imaging also revealed that cells under 3D culture condition maintain increased autophagy over monolayer cultures. High-throughput adaptability of the system extends its application for the screening of compounds that cause caspase activation, autophagy, or both demonstrating the potential utility of the sensor probe for diverse biological applications.

Telomerase inhibitors induce mitochondrial oxidation and DNA damage-dependent cell death rescued by Bcl-2/Bcl-xL.

BACKGROUND: Reactivation of telomerase is a hallmark of cancer and the majority of cancers over-express telomerase. Telomerase-dependent telomere length maintenance confers immortality to cancer cells. However, telomere length-independent cell survival functions of telomerase also play a critical role in tumorigenesis. Multiple telomerase inhibitors have been developed as therapeutics and include anti-sense oligonucleotides, telomerase RNA component targeting agents, chemical inhibitors of telomerase, small molecule inhibitors of hTERT, and telomerase vaccine. In general, telomerase inhibitors affect cell proliferation and survival of cells depending on the telomere length reduction, culminating in replicative senescence or cell death by crisis. However, most telomerase inhibitors kill cancer cells prior to significant reduction in telomere length, suggesting telomere length independent role of telomerase in early telomere dysfunction-dependent cell death. METHODS: In this study, we explored the mechanism of cell death induced by three prominent telomerase inhibitors utilizing a series of genetically encoded sensor cells including redox and DNA damage sensor cells. RESULTS: We report that telomerase inhibitors induce early cell cycle inhibition, followed by redox alterations at cytosol and mitochondria. Massive mitochondrial oxidation and DNA damage induce classical cell death involving mitochondrial transmembrane potential loss and mitochondrial permeabilization. Real-time imaging of the progression of mitochondrial oxidation revealed that treated cells undergo a biphasic mitochondrial redox alteration during telomerase inhibition, emphasizing the potential role of telomerase in the redox regulation at mitochondria. Additionally, silencing of hTERT confirmed its predominant role in maintaining mitochondrial redox homeostasis. Interestingly, the study also demonstrated that anti-apoptotic Bcl-2 family proteins still confer protection against cell death induced by telomerase inhibitors. CONCLUSION: The study demonstrates that redox alterations and DNA damage contribute to early cell death by telomerase inhibitors and anti-apoptotic Bcl-2 family proteins confer protection from cell death by their ability to safeguard mitochondria from oxidation damage.

Metastasis-associated protein 1 drives tumor cell migration and invasion through transcriptional repression of RING finger protein 144A.

Metastasis-associated protein 1 (MTA1), a component of the nucleosome-remodeling and histone deacetylase complex, is widely up-regulated in human cancers and significantly correlated with tumor invasion and metastasis, but the mechanisms involved remain largely unknown. Here, we report that MTA1 transcriptionally represses the expression of RING finger protein 144A (RNF144A), an uncharacterized gene whose protein product possesses potential E3 ubiquitin ligase activity, by recruiting the histone deacetylase 2 (HDAC2) and CCAAT/enhancer-binding protein α (c/EBPα) co-repressor complex onto human RNF144A promoter. Furthermore, an inverse correlation between the expression levels of MTA1 and RNF144A was demonstrated in publicly available breast cancer microarray datasets and the MCF10 breast cancer progression model system. To address functional aspects of MTA1 regulation of RNF144A, we demonstrate that RNF144A is a novel suppressor of cancer migration and invasion, two requisite steps of metastasis in vivo, and knockdown of endogenous RNF144A by small interfering RNAs accelerates the migration and invasion of MTA1-overexpressing cells. These results suggest that RNF144A is partially responsible for MTA1-mediated migration and invasion and that MTA1 overexpression in highly metastatic cancer cells drives cell migration and invasion by, at least in part, interfering with the suppressive function of RNF144A through transcriptional repression of RNF144A expression. Together, these findings provide novel mechanistic insights into regulation of tumor progression and metastasis by MTA1 and highlight a previously unrecognized role of RNF144A in MTA1-driven cancer cell migration and invasion.

Mechanism of MTA1 protein overexpression-linked invasion: MTA1 regulation of hyaluronan-mediated motility receptor (HMMR) expression and function.

Even though the hyaluronan-mediated motility receptor (HMMR), a cell surface oncogenic protein, is widely up-regulated in human cancers and correlates well with cell motility and invasion, the underlying molecular and nature of its putative upstream regulation remain unknown. Here, we found for the first time that MTA1 (metastatic tumor antigen 1), a master chromatin modifier, regulates the expression of HMMR and, consequently, its function in breast cancer cell motility and invasiveness. We recognized a positive correlation between the levels of MTA1 and HMMR in human cancer. Furthermore, MTA1 is required for optimal expression of HMMR. The underlying mechanism includes interaction of the MTA1·RNA polymerase II·c-Jun coactivator complex with the HMMR promoter to stimulates its transcription. Accordingly, selective siRNA-mediated knockdown of HMMR in breast cancer cells substantially reduces the invasion and migration of cells. These findings reveal a regulatory role for MTA1 as an upstream coactivator of HMMR expression and resulting biological phenotypes.

Real-time simultaneous imaging of temporal alterations in cytoplasmic and mitochondrial redox in single cells during cell division and cell death.

Cytosolic and organelle redox are highly interrelated, and their alterations play critical roles in both physiological and pathological cell states. This highly regulated process is crucial in life-death decisions of cells. Among organelles, the mitochondrion is the major source of intracellular-ROS and contributes to oxidation damage-induced cell death. Increase in cytosolic-redox and mitochondrial-redox is evident in cells undergoing diverse forms of cell death, such as apoptosis, necrosis, and necroptosis. The hierarchical profiling of redox signaling at the cytosol and mitochondria in a single cell is important to understand the relative contribution of each species in the initiation and shaping of cell death. Here, we demonstrate the potential application of ratiometric redox GFP (roGFP) and intensity-based redox-sensitive RFP (rxRFP) targeted to mitochondria in revealing both rapid and slow progressing changes in redox during cell division and in cells undergoing multiple modes of cell death. To generate imaging quality signal, single-cell clones stably expressing both roGFP at the cytosol and rxRFP probes targeted to mitochondria were generated. The cells provided sufficient temporal resolution with imaging-ready signal for the real-time visualization of rapidly progressing redox alterations at the cytosol and mitochondria. The long-time imaging of the cells revealed that a moderate increase in cytosolic ROS marks the division stage. Similarly, distinct forms of cell death trigger a unique and temporally regulated redox change at the cytosol and mitochondria, suggesting the potential utility of the sensor cells to dissect the nature of cell death pathways induced by specific forms of stress.

Endosomal-associated RFFL facilitates mitochondrial clearance by enhancing PRKN/parkin recruitment to mitochondria.

Mutations in the ubiquitin ligase PRKN (parkin RBR E3 ubiquitin protein ligase) are associated with Parkinson disease and defective mitophagy. Conceptually, PRKN-dependent mitophagy is classified into two phases: 1. PRKN recruits to and ubiquitinates mitochondrial proteins; 2. formation of phagophore membrane, sequestering mitochondria for degradation. Recently, endosomal machineries are reported to contribute to the later stage for membrane assembly. We reported a role for endosomes in the events upstream of phase 1. We demonstrate that the endosomal ubiquitin ligase RFFL (ring finger and FYVE like domain containing E3 ubiquitin protein ligase) associated with damaged mitochondria, and this association preceded that of PRKN. RFFL interacted with PRKN, and stable recruitment of PRKN to damaged mitochondria was substantially reduced in RFFL KO cells. Our study unraveled a novel role of endosomes in modulating upstream pathways of PRKN-dependent mitophagy initiation.Abbreviations CCCP: carbonyl cyanide 3-chlorophenylhydrazone; DMSO: dimethyl sulfoxide; EGFP: enhanced green fluorescence protein; KO: knockout; PRKN: parkin RBR E3 ubiquitin protein ligase; RFFL: ring finger and FYVE like domain containing E3 ubiquitin protein ligase; UQCRC1: ubiquinol-cytochrome c reductase core protein 1; WT: wild-type.

Quantitative Analysis of Apoptosis and Necrosis in Live Cells Using Flow Cytometry.

Apoptosis and necrosis are the two sides of the cell death penumbra. Apoptosis is a well-studied model of cell death wherein the cell destroys itself employing a predefined form of active signaling without the release of soluble cytoplasmic contents to the external environment. Compared to apoptosis, necrosis is a nonspecific form of sudden cell death in response to an invasive external stimulus which in turn is devoid of active programmed intracellular signaling leading to the sudden release of the soluble cellular contents consequent to the rupture of the cell membrane. This fundamental difference between apoptosis and necrosis made us believe that the former is the safe form of cell death and the latter is an undesirable one which often elicits an inflammatory response to the adjacent cells. Recent studies have shown that necrosis also involves a few defined cellular and complex biochemical events similar to apoptosis rendering it difficult to distinguish these two events at the single-cell level using the currently used popular assays.Here we provide a newly described detailed methodology encompassing cell system development along with a multiparametric flow cytometry-based approach to discriminate apoptotic cells from necrotic cells using a stable cell line expressing genetically encoded probe for detecting caspase activation and DsRed targeted at the mitochondria.

Evidence of a dysregulated vitamin D endocrine system in SARS-CoV-2 infected patient's lung cells.

Although a defective vitamin D endocrine system has been widely suspected to be associated in SARS-CoV-2 pathobiology, the status of the vitamin D endocrine system and vitamin D-modulated genes in lung cells of patients infected with SARS-CoV-2 remains unknown. To understand the significance of the vitamin D endocrine system in SARS-CoV-2 pathobiology, computational approaches were applied to transcriptomic datasets from bronchoalveolar lavage fluid (BALF) cells of such patients or healthy individuals. Levels of vitamin D receptor, retinoid X receptor, and CYP27A1 in BALF cells of patients infected with SARS-CoV-2 were found to be reduced. Additionally, 107 differentially expressed, predominantly downregulated genes, as potentially modulated by vitamin D endocrine system, were identified in transcriptomic datasets from patient's cells. Further analysis of differentially expressed genes provided eight novel genes with a conserved motif with vitamin D-responsive elements, implying the role of both direct and indirect mechanisms of gene expression by the dysregulated vitamin D endocrine system in SARS-CoV-2-infected cells. Protein-protein interaction network of differentially expressed vitamin D-modulated genes were enriched in the immune system, NF-κB/cytokine signaling, and cell cycle regulation as top predicted pathways that might be affected in the cells of such patients. In brief, the results presented here povide computational evidence to implicate a dysregulated vitamin D endocrine system in the pathobiology of SARS-CoV-2 infection.

Geometry encoded functional programming of tumor homing peptides for targeted drug delivery.

Poly-peptide molecules have shown promising applications in drug delivery and tumor targeting. A series of tumor homing peptides were designed by exhaustively sampling low energy geometrical basins of amino acids at specific sites of a peptide molecule to induce a conformational lock. This peptide library was pruned to a limited set of eight molecules, employing electrostatic interactions, docking, and molecular dynamics simulations. These designed and optimized peptides were synthesized and tested on various cell lines, including breast cancer (MDA-MB-231), cervical cancer (HeLa), osteosarcoma (U2-OS), and non-cancerous mammary epithelial cells (MCF-10A) using confocal microscopy and flow cytometry. Peptides show differential uptake in cancerous MDA-MB-231, HeLa, U2-OS, and non-cancerous MCF-10A cells. Confocal imaging verified their ability to penetrate even in 3D tumorospheres of MDA-MB-231 cells. Further, experiments of mitochondrial membrane potential depolarization and Caspase-3 activation confirmed that their cytotoxic effects are by apoptosis. Homing ability of the designed peptides in in vivo system and fluorescence imaging with clinical samples of human origin have further confirmed that the in vitro studies are qualitatively identical and quantitatively comparable in their ability to selectively recognize tumor cells. Overall, we present a roadmap for the functional programming of peptide-based homing and penetrating molecules that can perform selective tumor targeting.

Mitochondria targeted redox GFP reveals time and dose dependent onset and progression of mitochondrial oxidation with diverging cell death decisions during photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) is a successful cancer treatment modality. In vitro, in vivo, and clinical studies with different photosensitizers reveal diverging cell fates, including apoptosis, necrosis, autophagy, and non-specific forms of cell death. The mode of action and efficacy of PDT is mediated through free radical generation and is highly dependent on diverse variables such as nature, dose, metabolism of photosensitizer, irradiation energy, and irradiation cycle. AIM: Discovery of newer photosensitizers and optimization of PDT approaches to achieve a clinically relevant form of cell death called apoptosis requires better in vitro real-time methods. Oxidative damage and mitochondrial permeabilization are critical signaling events involved in photodamage and apoptosis. Hence, mitochondrial damage detection is an appropriate target signaling for mechanistic evaluation of PDT. METHODOLOGY: We report mitochondria-targeted redox GFP expressing cells as a sensitive system to test and validate important variables of PDT using the photosensitizer 5-Aminolevulinic acid (5-ALA) as a model. An independent FRET-based caspase sensor cell was also used to study the impact of the photosensitizer dosage and irradiation duration on the mode of cell death. RESULTS: The study reveals that the cancer cells expressing mt-roGFP are extremely sensitive to monitor mitochondrial oxidation induced by PDT. The extent of mitochondrial redox changes induced by PDT can be determined using these sensor cells by real-time image-based approaches. These approaches provide sufficient temporal resolution that is required to fine-tune and optimize the PDT conditions. The degree of oxidation of the probe is highly dependent on the dosage of photosensitizer and duration of light irradiation, which determines the nature of cell death. A real-time caspase sensor probe further confirmed that the caspase-dependent and caspase-independent nature of cell death is in high correlation with the extent of mitochondrial oxidation. A condition that triggers rapid and extreme mito-oxidation seems to favor necrosis, while delayed and slowly progressing redox changes contribute to caspase-dependent apoptosis. CONCLUSION: The study confirms that temporal analysis of mitochondrial oxidation is a reliable biomarker for fine-tuning PDT conditions to achieve the desired outcome. This can be achieved using stable cancer cell lines expressing mitochondria-targeted roGFP by ratiometric imaging.

A reporter cell line for real-time imaging of autophagy and apoptosis.

Simultaneous detection of autophagy and apoptosis is important in drug discovery and signaling studies. Here we report, a real-time reporter cell line for the simultaneous detection of apoptosis and autophagy at single-cell level employing stable integration of two fluorescent protein reporters of apoptosis and autophagy. Cells stably expressing EGFP-LC3 fusion was developed initially as a marker for autophagy and subsequently stably expressed with inter-mitochondrial membrane protein SMAC with RFP fusion to detect mitochondrial permeabilization event of apoptosis. The cell lines faithfully reported the LC3 punctae formation and release of intermembrane proteins in response to diverse apoptotic and autophagic stimuli.

Water-Templated, Polysaccharide-rich Bioartificial 3D Microarchitectures as Extra-Cellular Matrix Bioautomatons.

This is the first report of exploiting the "quasi-spherical" shape of water molecules for recapitulating a true human extracellular matrix (ECM). Herein, water behaved as a quasi-spherical porogen, for engineering polysaccharide-rich and chemically defined 3D-microarchitecture, with semi-interpenetrating networks (S-IPNs). Furthermore, their viscoelastic behavior along with a heterogeneous, fibroporous morphology, facilitated instructive, self-remodeling of the bioartificial scaffolds, thence effectively permitting and promoting the growth of 3D tumor spheroids of divergent origins. The hybrid composites displayed reproducible, uniform tumor spheroids with a Z-depth of ∼65 ± 2 µm in case of human adenocarcinoma (DLD-1) and ∼54 ± 3 µm for human glioblastoma cells (U-251) (vs. nonuniform spheroids, on Agarose matrix). Thereafter, their capacity for anticancer drug screening was examined using limited cancer drugs. The conflicting drug screening results for Etoposide's reduced efficacy on glioblastoma cells cultured on our 3D matrix could be ascribed to decreased drug access and thus lower ingression. Nonetheless, adenocarcinoma's resistance to Camptothecin was paralleled. Moreover, their potential for real-time, high-content, phenotypic precision oncology was affirmed by the exceptional transparency of the synthesized composite. Since this 3D microarchitecture typifies ECM bioautomaton, this matrix can also be wielded for precision oncology.

Syndiotactic peptides for targeted delivery.

Lack of cell-type specificity and proteolytic susceptibility have long been the major bottlenecks for the development of peptide-based biomaterials for targeted drug delivery. Though a poly-l backbone provides the adaptability to re-conform the peptide structure to bind to a receptor, it also makes the peptide more susceptible to proteolytic cleavage. We have attempted to address this issue by designing a set of syndiotactic peptides de novo, with alternating l- and d-amino acids in succession. The designed peptides have higher rates of cellular uptake than the Tat (48-60) peptide in breast and cervical cancer cells. The uptake is independent of concentration, temperature and endocytosis (clathrin mediated). Importantly, the peptides are stable in both human plasma and bovine serum. The peptide-drug conjugates are much less toxic to the non-cancerous cells than cancer cells. The designed peptides are a step forward towards the development of targeted drug delivery vectors on peptide templates. STATEMENT OF SIGNIFICANCE: Present options in chemotherapy have multiple side effects arising from the lack of cell-type specificity, which makes them synonymous with "a Pyrrhic victory". Proteolytic susceptibility and non-specificity towards cancer cells has stunted the development of peptide-based biomaterials for targeted drug delivery. We have designed a set of peptides, addressing the above-mentioned roadblocks at an in vitro level. The peptides were designed on the template of a naturally existing peptide antibiotic from Bacillus brevis. The designed peptides have higher rates of cellular transduction than the model peptide (Tat), and is majorly membrane based. The peptides are stable in serum and selective towards cancer cells. Observations presented in this work can potentially take the discipline of de novo design of biomaterial conjugates forward.

Molecular profiling of anastatic cancer cells: potential role of the nuclear export pathway.

PURPOSE: Anastasis is newly discovered process by which cells recover from late-stage apoptosis upon removal of a death stimulus. Recent reports suggest that cells may recover, even after the initiation of mitochondrial outer-membrane permeabilization (MOMP) and caspase activation. Here, we specifically studied the reversibility of late-stage apoptosis in cervical (HeLa) and breast (MDA-MB-231) cancer cells in relation to the extent of MOMP (limited or widespread). In addition, we explored the molecular factors involved in the anastatic process. METHODS: The extent of MOMP was assessed using time lapse confocal microscopic imaging, considering mitochondrial cytochrome c-GFP release as a marker for MOMP. Anastatic cells were generated by specifically recovering late-stage apoptotic (annexin V/PI positive) cervical and breast cancer cells. Molecular signaling events involved in death reversal were assessed using LC-MS/MS and qRT-PCR. Targeted chemical inhibition and shRNA-based gene silencing studies were employed to explore the role of the nuclear export pathway in anastasis and increased oncogenicity. RESULTS: Time-lapse imaging of drug-treated Cyt-c-GFP expressing cancer cells revealed cell recovery despite widespread MOMP. A few recovered anastatic cells were noted and these were found to proliferate through a selection-type of survival. They showed increased drug-resistance, migration and invasive potential compared to non-anastatic cancer cells. Network analysis using 49 proteins uniquely expressed in anastatic cells indicated upregulation of nuclear export/import, redox and Ras signaling pathways in both HeLa and MDA-MB-231 anastatic cells, indicating common molecular mechanisms in different cell types. Inhibition of XPO1 significantly reduced the recovery of apoptotic cells and abrogated acquired oncogenic transformation in the anastatic cancer cells. CONCLUSIONS: Our study indicates that cancer cells can revert from apoptosis even after the induction of widespread MOMP. We noted a significant role of the nuclear-export pathway in the anastatic process of cancer cells. Inhibition of anastasis through the nuclear export pathway may be a potential therapeutic strategy for targeting drug-resistance, metastasis and recurrence problems during cancer treatment.

A high-throughput real-time in vitro assay using mitochondrial targeted roGFP for screening of drugs targeting mitochondria.

Most toxic compounds including cancer drugs target mitochondria culminating in its permeabilization. Cancer drug-screening and toxicological testing of compounds require cost-effective and sensitive high-throughput methods to detect mitochondrial damage. Real-time methods for detection of mitochondrial damage are less toxic, allow kinetic measurements with good spatial resolution and are preferred over end-stage assays. Cancer cell lines stably expressing genetically encoded mitochondrial-targeted redox-GFP2 (mt-roGFP) were developed and validated for its suitability as a mitochondrial damage sensor. Diverse imaging platforms and flow-cytometry were utilized for ratiometric analysis of redox changes with known toxic and cancer drugs. Key events of cell death and mitochondrial damage were studied at single-cell level coupled with mt-roGFP. Cells stably expressing mt-roGFP and H2B-mCherry were developed for high-throughput screening (HTS) application. Most cancer drugs while inducing mitochondrial permeabilization trigger mitochondrial-oxidation that can be detected at single-cell level with mt-roGFP. The image-based assay using mt-roGFP outperformed other quantitative methods of apoptosis in ease of screening. Incorporation of H2B-mCherry ensures accurate and complete automated segmentation with excellent Z value. The results substantiate that most cancer drugs and known plant-derived antioxidants trigger cell-death through mitochondrial redox alterations with pronounced ratio change in the mt-roGFP probe. Real-time analysis of mitochondrial oxidation and mitochondrial permeabilization reveal a biphasic ratio change in dying cells, with an initial redox surge before mitochondrial permeabilization followed by a drastic increase in ratio after complete mitochondrial permeabilization. Overall, the results prove that mitochondrial oxidation is a reliable indicator of mitochondrial damage, which can be readily determined in live cells using mt-roGFP employing diverse imaging techniques. The assay described is highly sensitive, easy to adapt to HTS platforms and is a valuable resource for identifying cytotoxic agents that target mitochondria and also for dissecting cell signaling events relevant to redox biology.

A Real-Time Image-Based Approach to Distinguish and Discriminate Apoptosis from Necrosis.

Recent cell biology studies reveal that a cell can die through multiple pathways via distinct signaling mechanisms. Among these, apoptosis and necrosis are two distinct cell death pathways, and their detection and discrimination is vital in the drug discovery process and in understanding diverse biological processes. Although sensitive assays for apoptosis and necrosis are available, it is extremely difficult to adapt any of these methods to discriminate apoptosis-inducing stimuli from necrosis-inducing stimuli because of the acquisition of secondary necrosis by apoptotic cells when they are not phagocytosed. Essentially, any assay for discriminating apoptosis and necrosis needs to be carried out in real-time kinetic mode. Caspase 3 or 7 activation is observed in the majority of apoptotic cell death. Similarly, the absence of caspase 3/7 activation and cell membrane leakage are the two prominent indicators for necrotic cell death or necroptosis. The programmed form of necrosis, called pyroptosis, is also accompanied by membrane leakage and most often associated with activation of specific caspases such as caspase 1, 4, or 11, but not through caspase 3/7 activation. Here, a robust and sensitive real-time method is described to distinguish and discriminate apoptosis from necrosis. The assay utilizes stable integration of a genetically encoded fluorescence resonance energy transfer (FRET) probe for caspase 3/7 activation and the mitochondrion-targeted DsRed to identify necrotic cells. Caspase activation is determined by cleavage of the FRET probe; loss of soluble FRET probe with retention of mitochondrial red fluorescence indicates necrosis. This unit describes an important protocol for the generation of sensor cells expressing both probes, followed by detailed analysis of apoptosis and necrosis by microscopy imaging, confocal imaging, high-throughput imaging, and flow cytometry. © 2018 by John Wiley & Sons, Inc.