STAT3 (hEGF) Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of STAT3
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Signal transducers and activators of transcription (STAT) is a family (STAT 1, 2, 3, 4, 5a, 5b, and 6) of cytoplasmic proteins that function as secondary messengers and transcription factors. STAT proteins are activated via phosphorylation by Janus kinases (JAKs) in response to cytokine activation of cell surface receptor tyrosine kinases. Interestingly, epidermal growth factor (EGF) upon binding to the EGF receptor (EGFR), also activates STAT signalling pathways either dependently or independently of JAKs. Following STAT activation the proteins dimerize and localize to the nucleus where they activate transcription of a selected set of genes.

It is known that STAT3 regulates important functions such as embryogenesis, apoptosis, skin remodelling, cell migration as well as macrophage inactivation and down-regulation of inflammatory cytokines in T-helper-cell responses.

Figure 1. Translocation of EGFP-STAT3 stimulated with EGF in response to PD158780. Cells were treated with 100 ng/mL EGF in the absence (DMSO control, left panel) or presence (right panel) of 1 µM PD158780. Arrows indicate EGF-mediated nuclear translocation detected by the image analysis algorithm.

Figure 2: PD158780 concentration response curve in the STAT3 (hEGF) Redistribution assay (n=8). Concentration response was measured in 9 point half log dilution series of PD158780. Cells were then fixed and the nucleus to cytoplasm translocation was measured using the Cellomics ArrayScan® V^TI  Reader and the RedistributionV3 BioApplication. % activity was calculated relative to the positive (1 µM PD158780) and negative control (0.25% DMSO). The EC₅₀  value of PD158780 is approximately 107 nM in the assay.