19 results on '"Conos, Stephanie A"'
Search Results
2. Noncoding RNAs: Master Regulators of Inflammatory Signaling
- Author
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Chew, Chen Li, Conos, Stephanie Ana, Unal, Bilal, and Tergaonkar, Vinay
- Published
- 2018
- Full Text
- View/download PDF
3. Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner
- Author
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Conos, Stephanie A., Chen, Kaiwen W., De Nardo, Dominic, Hara, Hideki, Whitehead, Lachlan, Núñez, Gabriel, Masters, Seth L., Murphy, James M., Schroder, Kate, Vaux, David L., Lawlor, Kate E., Lindqvist, Lisa M., and Vince, James E.
- Published
- 2017
4. Pyroptosis
- Author
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Lawlor, Kate E., primary, Conos, Stephanie, additional, and Vince, James E., additional
- Published
- 2018
- Full Text
- View/download PDF
5. Divergent roles for caspase-8 and MLKL in high-fat diet induced obesity and NAFLD in mice
- Author
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Tye, Hazel, primary, Conos, Stephanie A, additional, Djajawi, Tirta M, additional, Abdoulkader, Nasteho, additional, Kong, Isabella Y, additional, Kaummon, Helene L, additional, Narayana, Vinod K, additional, Speir, Mary, additional, Gottschalk, Timothy A, additional, Emery, Jack, additional, Simpson, Daniel S, additional, Hall, Cathrine, additional, Vince, Angelina J, additional, Russo, Sophia, additional, Crawley, Rhiannan, additional, Rashidi, Maryam, additional, Hildebrand, Joanne, additional, Murphy, James M, additional, Whitehead, Lachlan, additional, De Souza, David P., additional, Masters, Seth L, additional, Hawkins, Edwin D, additional, Murphy, Andrew J, additional, Vince, James, additional, and Lawlor, Kate, additional
- Published
- 2023
- Full Text
- View/download PDF
6. Caspase‐8‐driven apoptotic and pyroptotic crosstalk causes cell death and IL ‐1β release in X‐linked inhibitor of apoptosis ( XIAP ) deficiency
- Author
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Hughes, Sebastian A, primary, Lin, Meng, additional, Weir, Ashley, additional, Huang, Bing, additional, Xiong, Liya, additional, Chua, Ngee Kiat, additional, Pang, Jiyi, additional, Santavanond, Jascinta P, additional, Tixeira, Rochelle, additional, Doerflinger, Marcel, additional, Deng, Yexuan, additional, Yu, Chien‐Hsiung, additional, Silke, Natasha, additional, Conos, Stephanie A, additional, Frank, Daniel, additional, Simpson, Daniel S, additional, Murphy, James M, additional, Lawlor, Kate E, additional, Pearson, Jaclyn S, additional, Silke, John, additional, Pellegrini, Marc, additional, Herold, Marco J, additional, Poon, Ivan K H, additional, Masters, Seth L, additional, Li, Mingsong, additional, Tang, Qin, additional, Zhang, Yuxia, additional, Rashidi, Maryam, additional, Geng, Lanlan, additional, and Vince, James E, additional
- Published
- 2023
- Full Text
- View/download PDF
7. Simultaneous Detection of Cellular Viability and Interleukin-1β Secretion from Single Cells by ELISpot
- Author
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Conos, Stephanie A., primary, Lindqvist, Lisa M., additional, and Vince, James E., additional
- Published
- 2017
- Full Text
- View/download PDF
8. Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway
- Author
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Simpson, Daniel S., primary, Pang, Jiyi, additional, Weir, Ashley, additional, Kong, Isabella Y., additional, Fritsch, Melanie, additional, Rashidi, Maryam, additional, Cooney, James P., additional, Davidson, Kathryn C., additional, Speir, Mary, additional, Djajawi, Tirta M., additional, Hughes, Sebastian, additional, Mackiewicz, Liana, additional, Dayton, Merle, additional, Anderton, Holly, additional, Doerflinger, Marcel, additional, Deng, Yexuan, additional, Huang, Allan Shuai, additional, Conos, Stephanie A., additional, Tye, Hazel, additional, Chow, Seong H., additional, Rahman, Arfatur, additional, Norton, Raymond S., additional, Naderer, Thomas, additional, Nicholson, Sandra E., additional, Burgio, Gaetan, additional, Man, Si Ming, additional, Groom, Joanna R., additional, Herold, Marco J., additional, Hawkins, Edwin D., additional, Lawlor, Kate E., additional, Strasser, Andreas, additional, Silke, John, additional, Pellegrini, Marc, additional, Kashkar, Hamid, additional, Feltham, Rebecca, additional, and Vince, James E., additional
- Published
- 2022
- Full Text
- View/download PDF
9. Targeting RIP Kinases in Chronic Inflammatory Disease
- Author
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Speir, Mary, primary, Djajawi, Tirta M., additional, Conos, Stephanie A., additional, Tye, Hazel, additional, and Lawlor, Kate E., additional
- Published
- 2021
- Full Text
- View/download PDF
10. XIAP Loss Triggers RIPK3- and Caspase-8-Driven IL-1β Activation and Cell Death as a Consequence of TLR-MyD88-Induced cIAP1-TRAF2 Degradation
- Author
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Lawlor, Kate E., primary, Feltham, Rebecca, additional, Yabal, Monica, additional, Conos, Stephanie A., additional, Chen, Kaiwen W., additional, Ziehe, Stephanie, additional, Graß, Carina, additional, Zhan, Yifan, additional, Nguyen, Tan A., additional, Hall, Cathrine, additional, Vince, Angelina J., additional, Chatfield, Simon M., additional, D’Silva, Damian B., additional, Pang, Kenneth C., additional, Schroder, Kate, additional, Silke, John, additional, Vaux, David L., additional, Jost, Philipp J., additional, and Vince, James E., additional
- Published
- 2017
- Full Text
- View/download PDF
11. A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation
- Author
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Conn, Vanessa M., primary, Hugouvieux, Véronique, additional, Nayak, Aditya, additional, Conos, Stephanie A., additional, Capovilla, Giovanna, additional, Cildir, Gökhan, additional, Jourdain, Agnès, additional, Tergaonkar, Vinay, additional, Schmid, Markus, additional, Zubieta, Chloe, additional, and Conn, Simon J., additional
- Published
- 2017
- Full Text
- View/download PDF
12. BAX-BAK1-independent LC3B lipidation by BH3 mimetics is unrelated to BH3 mimetic activity and has only minimal effects on autophagic flux
- Author
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Reljic, Boris, primary, Conos, Stephanie, additional, Lee, Erinna F., additional, Garnier, Jean-Marc, additional, Dong, Li, additional, Lessene, Guillaume, additional, Fairlie, W. Douglas, additional, Vaux, David L., additional, and Lindqvist, Lisa M., additional
- Published
- 2016
- Full Text
- View/download PDF
13. XIAP Loss Triggers RIPK3- and Caspase-8-Driven IL-1β Activation and Cell Death as a Consequence of TLR-MyD88-Induced cIAP1-TRAF2 Degradation
- Author
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Philipp J. Jost, Angelina J. Vince, Stephanie A. Conos, Yifan Zhan, Kate Schroder, David L. Vaux, Simon M Chatfield, Monica Yabal, Carina Graß, Stephanie Ziehe, John Silke, Kaiwen W. Chen, Kate E. Lawlor, Ken C Pang, Tan A. Nguyen, James E Vince, Damian B D'Silva, Rebecca Feltham, Cathrine Hall, Lawlor, Kate E, Feltham, Rebecca, Yabal, Monica, Conos, Stephanie A, Chen, Kaiwen W, Ziehe, Stephanie, Graß, Carina, Zhan, Yifan, Nguyen, Tan A, Hall, Cathrine, Vince, Angelina J, Chatfield, Simon M, D'Silva, Damian B, Pang, Kenneth C, Schroder, Kate, Silke, John, Vaux, David L, Jost, Philipp J, and Vince, James E
- Subjects
0301 basic medicine ,Programmed cell death ,TRAF2 ,proteasomal degradation ,Necroptosis ,Interleukin-1beta ,necroptosis ,Inhibitor of apoptosis ,Caspase 8 ,RIPK3 ,General Biochemistry, Genetics and Molecular Biology ,caspase-8 ,Inhibitor of Apoptosis Proteins ,03 medical and health sciences ,Mice ,XIAP ,NLRP3 ,autoinflammatory disease ,autoinflammatory conditions ,Toll-like receptor ,medicine ,XIAP deficiency ,Animals ,XIAP Deficiency ,lcsh:QH301-705.5 ,Mice, Knockout ,Cell Death ,Chemistry ,Toll-Like Receptors ,apoptosis ,Inflammasome ,interferon ,TNF Receptor-Associated Factor 2 ,3. Good health ,cIAP1 ,TNFR2 ,030104 developmental biology ,cell death ,interleukin (IL)-18 ,lcsh:Biology (General) ,Receptor-Interacting Protein Serine-Threonine Kinases ,Myeloid Differentiation Factor 88 ,Proteolysis ,Cancer research ,medicine.drug - Abstract
X-linked Inhibitor of Apoptosis (XIAP) deficiency predisposes people to pathogen-associated hyperinflammation. Upon XIAP loss, Toll-like receptor (TLR) ligation triggers RIPK3-caspase-8-mediated IL-1β activation and death in myeloid cells. How XIAP suppresses these events remains unclear. Here, we show that TLR-MyD88 causes the proteasomal degradation of the related IAP, cIAP1, and its adaptor, TRAF2, by inducing TNF and TNF Receptor 2 (TNFR2) signaling. Genetically, we define that myeloid-specific cIAP1 loss promotes TLR-induced RIPK3-caspase-8 and IL-1β activity in the absence of XIAP. Importantly, deletion of TNFR2 in XIAP-deficient cells limited TLR-MyD88-induced cIAP1-TRAF2 degradation, cell death, and IL-1β activation. In contrast to TLR-MyD88, TLR-TRIF-induced interferon (IFN)β inhibited cIAP1 loss and consequent cell death. These data reveal how, upon XIAP deficiency, a TLR-TNF-TNFR2 axis drives cIAP1-TRAF2 degradation to allow TLR or TNFR1 activation of RIPK3-caspase-8 and IL-1β. This mechanism may explain why XIAP-deficient patients can exhibit symptoms reminiscent of patients with activating inflammasome mutations. Refereed/Peer-reviewed
- Published
- 2017
14. Simultaneous Detection of Cellular Viability and Interleukin-1β Secretion from Single Cells by ELISpot
- Author
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Lisa M Lindqvist, Stephanie A. Conos, James E Vince, Conos, Stephanie A, Lindqvist, Lisa M, and Vince, James E
- Subjects
0301 basic medicine ,Programmed cell death ,biology ,interleukin-1β ,Chemistry ,pyroptosis ,ELISPOT ,ELISpot ,apoptosis ,caspase-1 ,Pyroptosis ,Caspase 1 ,caspase-8 ,Cell biology ,secretion ,03 medical and health sciences ,cell death ,030104 developmental biology ,inflammasome ,cytokine ,Extracellular ,biology.protein ,Secretion ,Viability assay ,Antibody - Abstract
Cell death results in the breakdown of the plasma membrane, which can cause the release of cytosolic proteins. During caspase-1-mediated cell death, termed pyroptosis, pro-inflammatory mediators that lack canonical secretory signal sequences, such as interleukin-1β (IL-1β), are released into the extracellular environment. To define whether cell death is required for the release of IL-1β, or if IL-1β can be actively secreted from viable cells, we have developed a modified IL-1β Enzyme-Linked ImmunoSpot (ELISpot) assay. This assay simultaneously detects cellular viability and IL-1β release at the single-cell level, and is therefore useful to examine how cell death influences IL-1β secretion under different experimental conditions. Cells expressing a surrogate viability marker, such as GFP, are plated onto cellulose filter plates coated with an IL-1β capture antibody. This antibody immobilizes IL-1β as it is released from cells, allowing detection of distinct IL-1β “spots.” Both GFP positive cells and IL-1β spots are detected and quantified using an AID ELISpot Reader, and the captured images are overlaid. Therefore, cell viability and IL-1β release from individual cells can be monitored visually. We have recently used this method to document how individual fibroblasts expressing activated caspase-1 can secrete IL-1β in the absence of cell death. Adaptation of this assay to other experimental conditions may help to define the circumstances where cell death influences IL-1β release and IL-1β-driven inflammatory responses. Refereed/Peer-reviewed
- Published
- 2017
15. Noncoding RNAs: Master Regulators of Inflammatory Signaling
- Author
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Bilal Unal, Vinay Tergaonkar, Chen Li Chew, Stephanie A. Conos, Chew, Chen Li, Conos, Stephanie Ana, Unal, Bilal, and Tergaonkar, Vinay
- Subjects
0301 basic medicine ,RNA, Untranslated ,MAP Kinase Signaling System ,Inflammation ,Computational biology ,Biology ,Transcriptome ,03 medical and health sciences ,medicine ,Animals ,Humans ,NF-KB signaling ,Molecular Biology ,Regulation of gene expression ,ERK/MAPK pathway ,NF-kappa B ,Non-coding RNA ,ncRNA ,Nf κb signaling ,030104 developmental biology ,Gene Expression Regulation ,inflammation ,IncRNA ,Molecular Medicine ,medicine.symptom ,Signal transduction ,Signal Transduction - Abstract
Inflammatory signaling underlies many diseases, from arthritis to cancer. Our understanding of inflammation has thus far been limited to the world of proteins, because we are only just beginning to understand the role that noncoding RNAs (ncRNA) might play. It is now clear that ncRNA do not constitute transcriptional 'noise' but instead harbor physiological functions in controlling signaling pathways. In this review, we cover the newly discovered mechanisms and functions of ncRNAs in the regulation of inflammatory signaling. We also describe advances in experimental techniques allowing this field of research to take root. These findings have opened new avenues for putative therapeutic intervention in inflammatory diseases, which may be seen translated into clinical outcomes in the future. Refereed/Peer-reviewed
- Published
- 2017
16. A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation
- Author
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Chloe Zubieta, Markus Schmid, Vanessa M. Conn, Aditya Nayak, Véronique Hugouvieux, Agnès Jourdain, Simon J. Conn, Gökhan Cildir, Giovanna Capovilla, Vinay Tergaonkar, Stephanie A. Conos, Centre for Cancer Biology, Hanson Institute, Adelaide, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Department of Molecular Biology [Tübingen], Max Planck Institute for Developmental Biology, Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Department of Biochemistry, Yong Loo Lin School of Medicine and Department of Biological Sciences, National University of Singapore (NUS), Laboratory of NF-κB Signaling, Institute of Molecular and Cell Biology, Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, ANR (FLOPINET), CNRS, NHMRC [GNT1089167], Australian Research Council [FT160100318], ATIP-Avenir, LabEx GRAL [ANR-10-LABX-49-01], South Australian Government Department of State Development, CEA Irtelis fellowship, ANR-16-CE92-0023,FLOPINET,Floral Pioneer Factors(2016), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Conn, Vanessa M, Hugouvieux, Véronique, Nayak, Aditya, Conos, Stephanie A, Capovilla, Giovanna, Cildir, Gökhan, Jourdain, Agnés, Tergaonkar, Vinay, Schmid, Markus, Zubieta, Chloe, Conn, Simon J, Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-16-CE92-0023,FLOPINET,Floral Pioneer Factors, and ANR-10-LABX-49-01,Labex GRAL
- Subjects
0106 biological sciences ,0301 basic medicine ,circular RNAs (circRNAs) ,DNA, Plant ,RNA Splicing ,SEP3 ,Arabidopsis ,Plant Science ,Biology ,01 natural sciences ,Flowering ,03 medical and health sciences ,Splicing factor ,Exon ,alternative splicing ,Circular RNA ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,RNA, Messenger ,Gene ,SEPALLATA3 ,Homeodomain Proteins ,Genetics ,Arabidopsis Proteins ,Alternative splicing ,RNA ,Plant ,Gene regulation ,030104 developmental biology ,back-splicing ,RNA splicing ,DNA, Circular ,Transcription factor ,Homeotic gene ,Transcription Factors ,010606 plant biology & botany - Abstract
Simon J. Conn (simon.conn@unisa.edu.au); International audience; Circular RNAs (circRNAs) are a diverse and abundant class of hyper-stable, non-canonical RNAs that arise through a form of alternative splicing (AS) called back-splicing. These single-stranded, covalently-closed circRNA molecules have been identified in all eukaryotic kingdoms of life(1), yet their functions have remained elusive. Here, we report that circRNAs can be used as $bona\ fide$ biomarkers of functional, exon-skipped AS variants in $Arabidopsis$, including in the homeotic MADS-box transcription factor family. Furthermore, we demonstrate that circRNAs derived from exon 6 of the SEPALLATA3 (SEP3) gene increase abundance of the cognate exon-skipped AS variant (SEP3.3 which lacks exon 6), in turn driving floral homeotic phenotypes. Toward demonstrating the underlying mechanism, we show that the SEP3 exon 6 circRNA can bind strongly to its cognate DNA locus, forming an RNA:DNA hybrid, or R-loop, whereas the linear RNA equivalent bound significantly more weakly to DNA. R-loop formation results in transcriptional pausing, which has been shown to coincide with splicing factor recruitment and AS(2-4). This report presents a novel mechanistic insight for how at least a subset of circRNAs probably contribute to increased splicing efficiency of their cognate exon-skipped messenger RNA and provides the first evidence of an organismal-level phenotype mediated by circRNA manipulation.
- Published
- 2017
17. Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner
- Author
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James M. Murphy, Lachlan Whitehead, Lisa M Lindqvist, Seth L. Masters, Stephanie A. Conos, Dominic De Nardo, Hideki Hara, Kate Schroder, James E Vince, Gabriel Núñez, Kate E. Lawlor, David L. Vaux, Kaiwen W. Chen, Conos, Stephanie A, Chen, Kaiwen W, De Nardo, Dominic, Hara, Hideki, Whitehead, Lachlan, Nunez, Gabriel, Masters, Seth L, Murphy, James M, Schroder, Kate, Vaux, David L, Lawlor, Kate E, Lindqvist, Lisa M, and Vince, James E
- Subjects
0301 basic medicine ,Programmed cell death ,Multidisciplinary ,integumentary system ,interleukin-1β ,Necroptosis ,Pyroptosis ,Caspase 1 ,necroptosis ,Signal transducing adaptor protein ,Inflammasome ,Biology ,Gasdermin D ,Proinflammatory cytokine ,Cell biology ,Multidisciplinary Sciences ,03 medical and health sciences ,030104 developmental biology ,NLRP3 ,medicine ,Secretion ,MLKL ,medicine.drug - Abstract
Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)- like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-Associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. diseases. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKLinduced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-Triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKLdependent Refereed/Peer-reviewed
- Published
- 2017
18. BAX-BAK1-independent LC3B lipidation by BH3 mimetics is unrelated to BH3 mimetic activity and has only minimal effects on autophagic flux
- Author
-
Erinna F. Lee, Boris Reljic, Jean-Marc Garnier, Guillaume Lessene, Lisa M Lindqvist, W. Douglas Fairlie, David L. Vaux, Stephanie A. Conos, Li Dong, Reljic, Boris, Conos, Stephanie, Lee, Erinna F, Garnier, Jean-Marc, Dong, Li, Lessene, Guillaume, Fairlie, W Douglas, Vaux, David L, and Lindqvist, Lisa M
- Subjects
0301 basic medicine ,autophagy ,BCL2 ,Programmed cell death ,Lipid-anchored protein ,Apoptosis ,Biology ,Piperazines ,Nitrophenols ,03 medical and health sciences ,Mice ,Bcl-2-associated X protein ,ABT-263 ,Autophagy ,Animals ,Molecular Biology ,Cells, Cultured ,bcl-2-Associated X Protein ,Sulfonamides ,Aniline Compounds ,ABT-737 ,fungi ,Biphenyl Compounds ,Basic Brief Report ,apoptosis ,BECN1 ,Cell Biology ,Bridged Bicyclo Compounds, Heterocyclic ,Cell biology ,Biphenyl compound ,cell death ,030104 developmental biology ,bcl-2 Homologous Antagonist-Killer Protein ,Proto-Oncogene Proteins c-bcl-2 ,ABT-199 ,biology.protein ,biological phenomena, cell phenomena, and immunity ,MAP1LC3B ,Microtubule-Associated Proteins ,Bcl-2 Homologous Antagonist-Killer Protein - Abstract
Inhibition of prosurvival BCL2 family members can induce autophagy, but the mechanism is controversial. We have provided genetic evidence that BCL2 family members block autophagy by inhibiting BAX and BAK1, but others have proposed they instead inhibit BECN1. Here we confirm that small molecule BH3 mimetics can induce BAX- and BAK1-independent MAP1LC3B/LC3B lipidation, but this only occurred at concentrations far greater than required to induce apoptosis and dissociate canonical BH3 domain-containing proteins that bind more tightly than BECN1. Because high concentrations of a less-active enantiomer of ABT-263 also induced BAX- and BAK1-independent LC3B lipidation, induction of this marker of autophagy appears to be an off-target effect. Indeed, robust autophagic flux was not induced by BH3 mimetic compounds in the absence of BAX and BAK1. Therefore at concentrations that are on target and achievable in vivo, BH3 mimetics only induce autophagy in a BAX- and BAK1-dependent manner. Refereed/Peer-reviewed
- Published
- 2016
19. Simultaneous Detection of Cellular Viability and Interleukin-1β Secretion from Single Cells by ELISpot.
- Author
-
Conos SA, Lindqvist LM, and Vince JE
- Subjects
- Animals, Cell Survival, Cells, Cultured, Mice, Embryo, Mammalian cytology, Embryo, Mammalian metabolism, Enzyme-Linked Immunosorbent Assay methods, Fibroblasts cytology, Fibroblasts metabolism, Interleukin-1beta metabolism, Single-Cell Analysis methods
- Abstract
Cell death results in the breakdown of the plasma membrane, which can cause the release of cytosolic proteins. During caspase-1-mediated cell death, termed pyroptosis, pro-inflammatory mediators that lack canonical secretory signal sequences, such as interleukin-1β (IL-1β), are released into the extracellular environment. To define whether cell death is required for the release of IL-1β, or if IL-1β can be actively secreted from viable cells, we have developed a modified IL-1β Enzyme-Linked ImmunoSpot (ELISpot) assay. This assay simultaneously detects cellular viability and IL-1β release at the single-cell level, and is therefore useful to examine how cell death influences IL-1β secretion under different experimental conditions. Cells expressing a surrogate viability marker, such as GFP, are plated onto cellulose filter plates coated with an IL-1β capture antibody. This antibody immobilizes IL-1β as it is released from cells, allowing detection of distinct IL-1β "spots." Both GFP positive cells and IL-1β spots are detected and quantified using an AID ELISpot Reader, and the captured images are overlaid. Therefore, cell viability and IL-1β release from individual cells can be monitored visually. We have recently used this method to document how individual fibroblasts expressing activated caspase-1 can secrete IL-1β in the absence of cell death. Adaptation of this assay to other experimental conditions may help to define the circumstances where cell death influences IL-1β release and IL-1β-driven inflammatory responses.
- Published
- 2018
- Full Text
- View/download PDF
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