Cargando…
HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway
Moderate environmental and physiological stressors are known to initiate protective heat shock response (HSR) leading to cell survival. HSR is largely mediated by the activation of heat shock factor (HSF), resulting in increased heat shock protein expression. Dysregulation of the HSR signaling has b...
Autores principales: | , , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
Bentham Open
2009
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2793398/ https://www.ncbi.nlm.nih.gov/pubmed/20161831 http://dx.doi.org/10.2174/1875397300903010001 |
_version_ | 1782175318251929600 |
---|---|
author | Hancock, Michael K Xia, Menghang Frey, Elizabeth S Sakamuru, Srilatha Bi, Kun |
author_facet | Hancock, Michael K Xia, Menghang Frey, Elizabeth S Sakamuru, Srilatha Bi, Kun |
author_sort | Hancock, Michael K |
collection | PubMed |
description | Moderate environmental and physiological stressors are known to initiate protective heat shock response (HSR) leading to cell survival. HSR is largely mediated by the activation of heat shock factor (HSF), resulting in increased heat shock protein expression. Dysregulation of the HSR signaling has been associated with various diseases including cancer, inflammation and neurodegenerative disorders. Compounds that can modulate HSR have been pursued for the treatment of these diseases. To facilitate the discovery of HSR modulators, we developed a high-throughput amenable betalactamase transcriptional reporter gene assay for monitoring the function of HSF. HeLa cells were engineered to express the beta-lactamase reporter under the control of HSF response elements (HSE) present in the HSP70 gene promoter. The HSE-beta lactamase (HSE-bla) reporter gene assay was validated by using HSF-specific siRNAs and known small molecule modulators. Taking the advantage of fluorescence resonance energy transfer (FRET)-based cell permeable betalactamase substrate, this assay can be miniaturized into 1536-well format. Our results demonstrate that the assay is robust and can be applied to high-throughput screening (HTS) for modulators of HSR. |
format | Text |
id | pubmed-2793398 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | Bentham Open |
record_format | MEDLINE/PubMed |
spelling | pubmed-27933982010-02-16 HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway Hancock, Michael K Xia, Menghang Frey, Elizabeth S Sakamuru, Srilatha Bi, Kun Curr Chem Genomics Article Moderate environmental and physiological stressors are known to initiate protective heat shock response (HSR) leading to cell survival. HSR is largely mediated by the activation of heat shock factor (HSF), resulting in increased heat shock protein expression. Dysregulation of the HSR signaling has been associated with various diseases including cancer, inflammation and neurodegenerative disorders. Compounds that can modulate HSR have been pursued for the treatment of these diseases. To facilitate the discovery of HSR modulators, we developed a high-throughput amenable betalactamase transcriptional reporter gene assay for monitoring the function of HSF. HeLa cells were engineered to express the beta-lactamase reporter under the control of HSF response elements (HSE) present in the HSP70 gene promoter. The HSE-beta lactamase (HSE-bla) reporter gene assay was validated by using HSF-specific siRNAs and known small molecule modulators. Taking the advantage of fluorescence resonance energy transfer (FRET)-based cell permeable betalactamase substrate, this assay can be miniaturized into 1536-well format. Our results demonstrate that the assay is robust and can be applied to high-throughput screening (HTS) for modulators of HSR. Bentham Open 2009-02-05 /pmc/articles/PMC2793398/ /pubmed/20161831 http://dx.doi.org/10.2174/1875397300903010001 Text en © Hancock et al.; Licensee Bentham Open. http://creativecommons.org/licenses/by-nc/3.0/ This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. |
spellingShingle | Article Hancock, Michael K Xia, Menghang Frey, Elizabeth S Sakamuru, Srilatha Bi, Kun HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway |
title | HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway |
title_full | HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway |
title_fullStr | HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway |
title_full_unstemmed | HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway |
title_short | HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway |
title_sort | hts-compatible β-lactamase transcriptional reporter gene assay for interrogating the heat shock response pathway |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2793398/ https://www.ncbi.nlm.nih.gov/pubmed/20161831 http://dx.doi.org/10.2174/1875397300903010001 |
work_keys_str_mv | AT hancockmichaelk htscompatibleblactamasetranscriptionalreportergeneassayforinterrogatingtheheatshockresponsepathway AT xiamenghang htscompatibleblactamasetranscriptionalreportergeneassayforinterrogatingtheheatshockresponsepathway AT freyelizabeths htscompatibleblactamasetranscriptionalreportergeneassayforinterrogatingtheheatshockresponsepathway AT sakamurusrilatha htscompatibleblactamasetranscriptionalreportergeneassayforinterrogatingtheheatshockresponsepathway AT bikun htscompatibleblactamasetranscriptionalreportergeneassayforinterrogatingtheheatshockresponsepathway |