A high-throughput, cell-based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the In Cell Western technique.
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.
Hoffman
Gregory R
GR
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. greg_hoffman@hms.harvard.edu
Moerke
Nathan J
NJ
Hsia
Max
M
Shamu
Caroline E
CE
Blenis
John
J
D000588:Amines / Q000737:chemistry / Q000528:radiation effects; D000918:Antibody Specificity; D015153:Blotting, Western; D002452:Cell Count; D002470:Cell Survival; D004353:Drug Evaluation, Preclinical; D023381:Endpoint Determination; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D007259:Infrared Rays; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D011506:Proteins; D034741:RNA, Small Interfering / Q000494:pharmacology*; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects*; D054852:Small Molecule Libraries; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000037:antagonists & inhibitors*; D014162:Transfection
D000588:Amines; D046912:Multiprotein Complexes; D011506:Proteins; D034741:RNA, Small Interfering; D054852:Small Molecule Libraries; D014157:Transcription Factors; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases