Real-time single cell analysis of Smac/DIABLO release during apoptosis

We examined the temporal and causal relationship between Smac/DIABLO release, cytochrome c (cyt-c) release, and caspase activation at the single cell level during apoptosis. Cells treated with the broad-spectrum caspase inhibitor z-VAD-fmk, caspase-3 (Casp-3)–deficient MCF-7 cells, as well as Bax-deficient DU-145 cells released Smac/DIABLO and cyt-c in response to proapoptotic agents. Real-time confocal imaging of MCF-7 cells stably expressing Smac/DIABLO-yellow fluorescent protein (YFP) revealed that the average duration of Smac/DIABLO-YFP release was greater than that of cyt-c-green fluorescent protein (GFP). However, there was no significant difference in the time to the onset of release, and both cyt-c-GFP and Smac/DIABLO-YFP release coincided with mitochondrial membrane potential depolarization. We also observed no significant differences in the Smac/DIABLO-YFP release kinetics when z-VAD-fmk–sensitive caspases were inhibited or Casp-3 was reintroduced. Simultaneous measurement of DEVDase activation and Smac/DIABLO-YFP release demonstrated that DEVDase activation occurred within 10 min of release, even in the absence of Casp-3.

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Ligand-Mediated Assembly and Real-Time Cellular Dynamics of Estrogen Receptor α-Coactivator Complexes in Living Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.13.4404-4412.2001 ◽

2001 ◽

Vol 21 (13) ◽

pp. 4404-4412 ◽

Cited By ~ 115

Author(s):

David L. Stenoien ◽

Anne C. Nye ◽

Maureen G. Mancini ◽

Kavita Patel ◽

Martin Dutertre ◽

...

Keyword(s):

Estrogen Receptor ◽

Real Time ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Steroid Receptor ◽

Estrogen Receptor Α ◽

Living Cells ◽

Live Cells ◽

Creb Binding Protein ◽

High Accumulation

ABSTRACT Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP–SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP–SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP–SRC-1, while antagonist additions diminish YFP–SRC-1–CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER–SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP–SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

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Glucose-dependent blood flow dynamics in murine pancreatic islets in vivo

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00715.2009 ◽

2010 ◽

Vol 298 (4) ◽

pp. E807-E814 ◽

Cited By ~ 49

Author(s):

Lara R. Nyman ◽

Eric Ford ◽

Alvin C. Powers ◽

David W. Piston

Keyword(s):

Blood Flow ◽

Blood Glucose ◽

Pancreatic Islets ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Cell Types ◽

Β Cells ◽

Islet Blood Flow ◽

Significant Difference

Pancreatic islets are highly vascularized and arranged so that regions containing β-cells are distinct from those containing other cell types. Although islet blood flow has been studied extensively, little is known about the dynamics of islet blood flow during hypoglycemia or hyperglycemia. To investigate changes in islet blood flow as a function of blood glucose level, we clamped blood glucose sequentially at hyperglycemic (∼300 mg/dl or 16.8 mM) and hypoglycemic (∼50 mg/dl or 2.8 mM) levels while simultaneously imaging intraislet blood flow in mouse models that express green fluorescent protein in the β-cells or yellow fluorescent protein in the α-cells. Using line scanning confocal microscopy, in vivo blood flow was assayed after intravenous injection of fluorescent dextran or sulforhodamine-labeled red blood cells. Regardless of the sequence of hypoglycemia and hyperglycemia, islet blood flow is faster during hyperglycemia, and apparent blood volume is greater during hyperglycemia than during hypoglycemia. However, there is no change in the order of perfusion of different islet endocrine cell types in hypoglycemia compared with hyperglycemia, with the islet core of β-cells usually perfused first. In contrast to the results in islets, there was no significant difference in flow rate in the exocrine pancreas during hyperglycemia compared with hypoglycemia. These results indicate that glucose differentially regulates blood flow in the pancreatic islet vasculature independently of blood flow in the rest of the pancreas.

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Investigation of the effect of Prunus Amygdalus Amara on the expression of some genes of apoptosis and immortality in breast cancer cells (MCF-7)

Current Drug Research Reviews ◽

10.2174/2589977513666211202094433 ◽

2021 ◽

Vol 13 ◽

Author(s):

Maryam Abdolahi-Majd ◽

Gholamhossein Hassanshahi ◽

Mahboubeh Vatanparast ◽

Mojgan Noroozi Karimabad

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Analysis ◽

Mrna Levels ◽

Prunus Amygdalus ◽

Mcf7 Cells ◽

Significant Difference ◽

Anti Cancer ◽

Bitter Almond ◽

Mcf 7

Background: Anti-cancer effects of almond nuts or oil have been approved, but there are a few pieces of research that have evaluated, in detail, almond and other seeds' effects on cancer. Therefore, in the present project, the aim was to explore the regulatory effect of the bitter almond extract (Prunus amygdalus Batsch) on the apoptotic and anti-cancer potency of MCF-7 cells. Objectives: In the current experimental research, the Almond effect on MCF7 cells was evaluated by investigating the expression and the balance between Bcl-2, Bax genes to unmark the potential molecular mechanism. Methods: For 24 and 48h, the MCF7 cells were treated with the bitter almond extract (187.5-3000 µg/mL). MTT assay was used to assess the viability, and Real-time-PCR was applied to determine the expression of Bax and Bcl-2, facing β-actin. Results: Our results revealed a significant difference between different extract concentrations on the viability of MCF7 cell lines in 24 and 48 h; cell viability decreased time-dependently (P < 0.05). After 24 and 48h of extract facing MCF7 cells, the evaluated IC50 value was 3000 and 1500 µg/mL, respectively. Based on Real Time-PCR analysis, after 24 and 48 h, the mRNA levels of BCL-2 decreased by the extract, whereas BAX was in the MCF-7 cell line. Conclusion: From the results, it can be concluded that bitter almond extract has anti-cancer properties that may influence the apoptotic pathways by regulating relative gene expression.

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A fate-mapping approach reveals the composite origin of the connecting tubule and alerts on “single-cell”-specific KO model of the distal nephron

AJP Renal Physiology ◽

10.1152/ajprenal.00286.2016 ◽

2016 ◽

Vol 311 (5) ◽

pp. F901-F906 ◽

Cited By ~ 22

Author(s):

Francesco Trepiccione ◽

Christelle Soukaseum ◽

Anna Iervolino ◽

Federica Petrillo ◽

Miriam Zacchia ◽

...

Keyword(s):

Single Cell ◽

Kidney Development ◽

Fluorescent Protein ◽

Collecting Duct ◽

Yellow Fluorescent Protein ◽

Cell Types ◽

Intercalated Cells ◽

Distal Nephron ◽

Principal Cells ◽

Different Cell Types

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl−cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

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High-Throughput, Long-Term Imaging of Salmonella Infecting Macrophages in a Micro-Environmental System

ASME 4th International Conference on Nanochannels, Microchannels, and Minichannels, Parts A and B ◽

10.1115/icnmm2006-96212 ◽

2006 ◽

Author(s):

Shih-Hui Chao ◽

Tim J. Strovas ◽

Ting-She M. Wang ◽

Kendan A. Jones-Isaac ◽

Susan L. Fink ◽

...

Keyword(s):

Single Cell ◽

Real Time ◽

Laser Scanning ◽

Single Cell Analysis ◽

Cell Analysis ◽

Cellular Functions ◽

Laser Scanning Confocal Microscope ◽

History Of ◽

Multiple Variables

Real-time single cell analysis is necessary to understand dynamic cellular functions in time and space. Such analyses require the simultaneous measurement of multiple variables in real-time, due to heterogeneity in cellular populations. We report the application of using a micro-environmental chamber on an automatic laser scanning confocal microscope to observe murine macrophage cells in incubation conditions for more than 18 hours. The motorized stage of the microscope was programmed to scan through pre-defined monitoring locations to increase the observation throughput. The acquired images were post-processed to extract the information of each cell. In contrast to current single-cell technologies, such as fluorescence-activated cell sorter (FACS) based systems, the reported architecture records the history of the physiological responses of individual cells.

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Real Time Single Cell Analysis of Bid Cleavage and Bid Translocation during Caspase-dependent and Neuronal Caspase-independent Apoptosis

Journal of Biological Chemistry ◽

10.1074/jbc.m511562200 ◽

2005 ◽

Vol 281 (9) ◽

pp. 5837-5844 ◽

Cited By ~ 56

Author(s):

Manus W. Ward ◽

Markus Rehm ◽

Heiko Duessmann ◽

Slavomir Kacmar ◽

Caoimhin G. Concannon ◽

...

Keyword(s):

Single Cell ◽

Real Time ◽

Single Cell Analysis ◽

Cell Analysis

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Calpain-mediated pathway dominates cisplatin-induced apoptosis in human lung adenocarcinoma cells as determined by real-time single cell analysis

International Journal of Cancer ◽

10.1002/ijc.23378 ◽

2008 ◽

Vol 122 (10) ◽

pp. 2210-2222 ◽

Cited By ~ 36

Author(s):

Lei Liu ◽

Da Xing ◽

Wei R. Chen ◽

Tongsheng Chen ◽

Yihui Pei ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Single Cell ◽

Real Time ◽

Human Lung ◽

Single Cell Analysis ◽

Cell Analysis ◽

Adenocarcinoma Cells ◽

Human Lung Adenocarcinoma Cells ◽

Lung Adenocarcinoma Cells ◽

Induced Apoptosis

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NEUROPHYSIOLOGICAL AND BEHAVIOURAL PERTURBATIONS IN Caenorhabditis elegans EXPOSED TO ORGANOPHOSPHATE PESTICIDES

Journal of Experimental Biology and Agricultural Sciences ◽

10.18006/2021.9(3).343.352 ◽

2021 ◽

Vol 9 (3) ◽

pp. 343-352

Author(s):

Rajul Jain ◽

◽

Priyanka Gautam ◽

Keyword(s):

Caenorhabditis Elegans ◽

Fluorescent Protein ◽

Developmental Toxicity ◽

Yellow Fluorescent Protein ◽

Model Organism ◽

Egg Laying ◽

High Exposure ◽

C Elegans ◽

Significant Difference ◽

Green Fluorescent

The ubiquitous use of pesticides all over the world leads to adverse effects on both targets as well as non-target species. The extensive and uncontrolled use of organophosphates (OPs), a large group of pesticidal compounds in agricultural and household products are resulting in high exposure to humans. This research has been carried out to study the adverse effect of OPs i.e., chlorpyrifos, trichlorfon, and disulfoton on model organism Caenorhabditis elegans to evaluate their behavioural as well as developmental toxicity at different time intervals i.e., 4, 24, 48, and 72 hours (hrs) of exposure. A significant difference was observed in all the behavioural endpoints like locomotion, egg-laying, offspring count, and learning along with developmental parameters like mortality, paralysis, and growth rendering from moderate to high toxic effects. Based on the above screening, trichlorfon resulted in glutamatergic and cholinergic neurodegeneration along with elevated autofluorescence. Loss in Yellow fluorescent Protein (YFP) and Green Fluorescent Protein (GFP) was recorded by 57.96% and 30.52% using transgenic strains OH11124 (otIs388 [eat-4(fosmid)::SL2::YFP::H2B + (pBX)pha-1(+)] III) and OH13083 (otIs576 [unc-17(fosmid)::GFP + lin-44::YFP]). These results have shown the biological potency of toxicants in C. elegans and pave the way forward to provide insight into various neurogenerative diseases in humans.

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