Fluorescence Lifetime Imaging of a Caspase‐3 Apoptosis Reporter

Buschhaus, Johanna M.; Gibbons, Anne E.; Luker, Gary D.

doi:10.1002/cpcb.36

Cited by 13 publications

(9 citation statements)

References 18 publications

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“…Here, we used FLIM to monitor a caspase-3 FRET imaging reporter to quantify apoptosis in in vitro and in vivo models [ 10 , 11 , 12 , 13 , 14 ]. The specifics of the FRET pair, LSS-mOrabge and mKate2, have been previously described [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis

Buschhaus

Humphries

Luker

2018

Cells

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Fluorescence lifetime imaging (FLIM) is a powerful imaging modality used to gather fluorescent reporter data independent of intracellular reporter intensity or imaging depth. We applied this technique to image real-time activation of a reporter for the proteolytic enzyme, caspase-3, in response to apoptotic cell death. This caspase-3 reporter activity provides valuable insight into cancer cell responsiveness to therapy and overall viability at a single-cell scale. Cleavage of a aspartate-glutamate-valine-aspartate (DEVD) linkage sequence alters Förster resonance energy transfer (FRET) within the reporter, affecting its lifetime. Cellular apoptosis was quantified in multiple environments ranging from 2D flat and 3D spheroid cell culture systems to in vivo murine mammary tumor xenografts. We evaluated cell-by-cell apoptotic responses to multiple pharmacological and genetic methods of interest involved in cancer cell death. Within this article, we describe methods for measuring caspase-3 activation at single-cell resolution in various complex environments using FLIM.

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Section: Introductionmentioning

confidence: 99%

A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis

Buschhaus

Humphries

Luker

2018

Cells

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“…To explore the function of overexpression of MIAT in IA, vascular endothelial cells were infected with oe-MIAT, which resulted in increased expression of cleaved Caspase 3, cleaved PARP1, and Bax along with decreased expression of Bcl-2. Moreover, Caspase-3, cleaved PARP1 (a substrate of caspase-3) and Bax (a Bcl-2 family protein) have been confirmed as vital indicators of apoptosis ( Bressenot et al, 2009 ; Buschhaus et al, 2017 ; Ling et al, 2017 ; Reyna et al, 2017 ). In addition, MIAT silencing could evidently reduce cell apoptosis, which was conducive to maintaining the continuity of endothelial cells and ultimately had a preventive effect on the occurrence of IA in this study.…”

Section: Discussionmentioning

confidence: 99%

Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

Zhao

Liu

et al. 2021

Front. Physiol.

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Intracranial aneurysm (IA) is vascular enlargement occurred on the wall of cerebral vessels and can result in fatal subarachnoid hemorrhage when ruptured. Recent studies have supported the important role of long non-coding RNAs (lncRNAs) in IA treatment. This study identified functional significance of lncRNA myocardial infarction associated transcript (MIAT) in IA. Myocardial infarction associated transcript and ectodermal-neural cortex 1 (ENC1) expression was detected by reverse transcription quantitative polymerase chain reaction. Cell counting kit 8 assay flow cytometry were conducted to detect cell viability and apoptosis of endothelial cells in IA. The interaction among MIAT, ENC1, and myelocytomatosis oncogene (MYC) was analyzed by RNA pull down, RNA immunoprecipitation assay, chromatin immunoprecipitation assay, and dual luciferase reporter assay. Intracranial aneurysm was induced by ligating the left carotid artery and the bilateral posterior branch of the renal artery in rats for studying the role of MIAT and ENC1 in vivo. Myocardial infarction associated transcript and ENC1 were upregulated in IA. Endothelial cells in IA presented a decreased cell viability and an increased apoptotic rate. Myocardial infarction associated transcript could regulate the expression of ENC1, and MYC could bind to the promoter region of ENC1. High expression of MIAT increased endothelial cell apoptosis and vascular endothelial injury, while MIAT knockdown was identified to reduce the risk of IA both in vitro and in vivo through regulating ENC1. To sum up, MIAT silencing is preventive for IA occurrence by decreasing the MYC-mediated ENC1 expression, which represents a novel therapeutic target for IA.

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“…The down-regulation of Bax and up-regulation of Bcl-2 induced by miR-208b overexpression are associated with the suppression of apoptosis under hypoxic conditions in MI [ 33 ]. Caspase-3 is an apoptotic signaling pathway-related protein that extensively participates in the induction of pyroptosis, a form of cell death [ 34 , 35 ]. Another study revealed that attenuated cardiomyocyte apoptosis was accompanied by a decreased cleaved caspase-3 expression, which consequently conferred the protection against cardiac injury [ 36 ].…”

Section: Discussionmentioning

confidence: 99%

Silencing long non-coding RNA MIAT ameliorates myocardial dysfunction induced by myocardial infarction via MIAT/miR-10a-5p/EGR2 axis

Cao

Ma²,

Wang

et al. 2021

Aging

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Long non-coding RNA (lncRNA) myocardial infarction-associated transcript (MIAT) has been widely-demonstrated to function as diagnostic markers for acute myocardial infarction (MI). This study was designed to explore the modulatory role of MIAT and its underlying molecular mechanism in MI. Firstly, MI mouse model was developed via ligation of the descending branch of the left coronary artery, and cell model was established through exposure to hypoxic conditions . Online prediction indicated that MIAT could bind to microRNA-10a-5p (miR-10a-5p), while miR-10a-5p was highlighted to bind to early growth response gene-2 (EGR2). MIAT and EGR2 were subsequently determined to be highly-expressed, whereas miR-10a-5p was found to be poorly-expressed in cardiomyocytes exposed to hypoxia as well as in MI mice using RT-qPCR and Western blot assay. The binding relationships between MIAT and miR-10a-5p, and between miR-10a-5p and EGR2 were further confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. The results of in vitro and in vivo experimentation also suggested that overexpression of miR-10a-5p or silencing of MIAT and EGR2 reduced cardiomyocyte apoptosis and increased ATP content, thus alleviating the impairment of cardiac function following MI. In a word, inhibition of MIAT protects against cardiac dysfunction induced by MI through the crosstalk with miR-10a-5p/EGR2.

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Fluorescence Lifetime Imaging of a Caspase‐3 Apoptosis Reporter

Cited by 13 publications

References 18 publications

A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis

A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis

Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

Silencing long non-coding RNA MIAT ameliorates myocardial dysfunction induced by myocardial infarction via MIAT/miR-10a-5p/EGR2 axis

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