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Updated: Jun 23, 2026
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Stimulation of Notch Signaling in Mouse Osteoclast Precursors
Published on: February 28, 2017
High-throughput screening compatible cell-based assay for interrogating activated notch signaling
Michael K Hancock1, Leisha Kopp, Kun Bi
1Invitrogen Corporation, Madison, Wisconsin 53719, USA.
Assay and Drug Development Technologies
|April 23, 2009
Summary
Aberrant Notch signaling is implicated in diseases, so we developed a cell-based assay to screen for pathway modulators. This assay identified novel compounds affecting Notch intracellular domain (NICD) signaling, including PI3K/AKT pathway involvement.
Area of Science:
- Cell Biology
- Molecular Biology
- Pharmacology
Background:
- Aberrant Notch pathway signaling is linked to developmental disorders, cancers, and neurodegenerative diseases.
- Modulating Notch signaling presents a therapeutic strategy for various diseases.
- The Notch pathway involves ligand binding, receptor cleavage, and nuclear translocation of Notch intracellular domain (NICD) to activate transcription.
Purpose of the Study:
- To develop and validate a cell-based assay for screening modulators of Notch intracellular domain (NICD) signaling.
- To identify novel compounds that can modulate NICD signaling.
- To investigate the role of the PI3K/AKT pathway in regulating NICD signaling.
Main Methods:
- Engineered HeLa cells with a Notch response element driving beta-lactamase (BLA) reporter gene expression.
- Utilized a tetracycline-inducible NICD expression system for controlled pathway activation.
- Screened 19 known compounds and employed RNA interference to validate modulators.
Main Results:
- The cell-based assay demonstrated a sensitive, quantitative readout for NICD signaling.
- Identified novel modulators of NICD signaling, including Wnt inhibitor ICG-001 which antagonized signaling.
- Histone deacetylase, HSP90, and PI3K/mTOR inhibitors (SAHA, 17-AAG, PI-103) activated NICD signaling.
- AKT and PI3K inhibitors (triciribine, GDC-0941) enhanced reporter activity, implicating the PI3K/AKT pathway.
Conclusions:
- The developed cell-based assay is suitable for high-throughput screening of NICD pathway modulators.
- The PI3K/AKT pathway plays a significant role in regulating NICD signaling.
- This system facilitates the discovery of potential therapeutic agents targeting the Notch pathway.
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