Fluorescence quantitation of thyrocyte iodide accumulation with the yellow fluorescent protein variant YFP-H148Q/I152L

2008 ◽  
Vol 373 (2) ◽  
pp. 239-246 ◽  
Author(s):  
Kerry J. Rhoden ◽  
Stefano Cianchetta ◽  
Serena Duchi ◽  
Giovanni Romeo
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Protein Variant

Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L

2007 ◽  
Vol 292 (2) ◽  
pp. C814-C823 ◽  
Author(s):  
Kerry J. Rhoden ◽  
Stefano Cianchetta ◽  
Valeria Stivani ◽  
Carla Portulano ◽  
Luis J. V. Galietta ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Sodium Iodide ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nuclear Imaging ◽  
Sodium Iodide Symporter ◽  
Protein Variant ◽  
Thyroid Cells

The sodium iodide symporter (NIS) mediates iodide (I−) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I−-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I− induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I− (35 μM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na+ dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I− influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I− influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I− also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I− uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I− may be a useful tool by which to study the pathophysiology and pharmacology of NIS.

Probing the excited state dynamics of Venus: origin of dual-emission in fluorescent proteins

2018 ◽  
Vol 207 ◽  
pp. 39-54 ◽  
Author(s):  
Shaina Dhamija ◽  
Bhisham Thakur ◽  
Purnananda Guptasarma ◽  
Arijit K. De
Keyword(s):  
Excited State ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Dual Emission ◽  
Protein Variant ◽  
Excited State Dynamics ◽  
Uv Excitation

We present studies on a yellow fluorescent protein variant, Venus, and investigate the photophysics behind the dual emission upon UV excitation in fluorescent proteins.

scholarly journals Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1

2021 ◽  
Vol 22 (13) ◽  
pp. 7100
Author(s):  
Yohan Seo ◽  
Sung Baek Jeong ◽  
Joo Han Woo ◽  
Oh-Bin Kwon ◽  
Sion Lee ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Cell ◽  
Channel Activity ◽  
Small Cell Lung ◽  
Protein Levels

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

scholarly journals The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3024
Author(s):  
Martin Fogtmann Berthelsen ◽  
Maria Riedel ◽  
Huiqiang Cai ◽  
Søren H. Skaarup ◽  
Aage K. O. Alstrup ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Somatic Cells ◽  
Genetic Alterations ◽  
Lung Epithelial Cells ◽  
Target Cells ◽  
Multiple Gene ◽  
Conditional Expression ◽  
Gene Alterations

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

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