Document Detail


A high-throughput, cell-based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the In Cell Western technique.
MedLine Citation:
PMID:  20085456     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The mTORC1 pathway is a central regulator of cell growth, and defective mTORC1 regulation plays a causative role in a variety of human diseases, including cancer, tumor syndromes such as the tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), and metabolic diseases such as diabetes and obesity. Given the importance of mTORC1 signaling in these diseases, there has been significant interest in developing screening methods suitable for identifying inhibitors of mTORC1 activation. To this end, we have developed a high-throughput, cell-based assay for the detection of rpS6-phosphorylation as a measure of mTORC1 signaling. This assay takes advantage of the "In Cell Western" (ICW) technique using the Aerius infrared imaging system (LI-COR Biosciences). The ICW procedure involves fixation and immunostaining of cells in a manner similar to standard immunofluorescence methods but takes advantage of secondary antibodies conjugated to infrared-excitable fluorophores for quantitative detection by the Aerius scanner. In addition, the cells are stained with an infrared-excitable succinimidyl ester dye, which covalently modifies free amine groups in fixed cells and provides a quantitative measure of cell number. We present validation data and pilot screens in a 384-well format demonstrating that this assay provides a statistically robust method for both small molecule and siRNA screening approaches designed to identify inhibitors of mTORC1 signaling.
Authors:
Gregory R Hoffman; Nathan J Moerke; Max Hsia; Caroline E Shamu; John Blenis
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Validation Studies    
Journal Detail:
Title:  Assay and drug development technologies     Volume:  8     ISSN:  1557-8127     ISO Abbreviation:  Assay Drug Dev Technol     Publication Date:  2010 Apr 
Date Detail:
Created Date:  2010-05-05     Completed Date:  2010-07-21     Revised Date:  2013-09-30    
Medline Journal Info:
Nlm Unique ID:  101151468     Medline TA:  Assay Drug Dev Technol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  186-99     Citation Subset:  IM    
Affiliation:
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. greg_hoffman@hms.harvard.edu
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MeSH Terms
Descriptor/Qualifier:
Amines / chemistry,  radiation effects
Antibody Specificity
Blotting, Western
Cell Count
Cell Survival
Drug Evaluation, Preclinical
Endpoint Determination
Fluorescent Antibody Technique, Indirect
HeLa Cells
Humans
Infrared Rays
Proteins
RNA, Small Interfering / pharmacology*
Reproducibility of Results
Signal Transduction / drug effects*
Small Molecule Libraries
Transcription Factors / antagonists & inhibitors*
Transfection
Grant Support
ID/Acronym/Agency:
CA46595/CA/NCI NIH HHS; GM51405/GM/NIGMS NIH HHS; R01 GM051405-16/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Amines; 0/Proteins; 0/RNA, Small Interfering; 0/Small Molecule Libraries; 0/Transcription Factors; 0/mechanistic target of rapamycin complex 1
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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