STAT3 (hIL-6) 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 transducer and activator of transcription 3 (STAT3) is involved in early embryogenesis, epithelial cell apoptosis, skin remodelling, and keratinocyte migration, as well as in macrophage inactivation and down regulation of inflammatory cytokines in T-helper-cell responses. STAT3 plays a central role in transmitting signals from the membrane to the nucleus, and binding of ligands such as the cytokine interleukin-6 (IL-6) to its receptor activates the Janus kinase (JAK)/STAT signalling pathway. Inactive STAT3 is cytoplasmic, but upon activation it is rapidly recruited to activated receptors, where its association with JAK catalyzes ligand-induced phosphorylation of STAT3. This leads to an SH2-mediated dimerization of STAT3, followed by translocation to the nucleus and activation of cytokine-responsive genes.

Figure 1. Translocation of EGFP-STAT3 stimulated with hIL-6 in response to JAK inhibitor 1. Cells were treated with 100 ng/mL hIL-6 in the absence (DMSO control, left panel) or presence (right panel) of 3 µM JAK inhibitor 1. Arrows indicate IL-6-mediated nuclear translocation detected by the image analysis algorithm.

Figure 2: JAK inhibtor 1 concentration response curve in the STAT3 (hIL-6) Redistribution assay (n=12). Concentration response was measured in 9 point half log dilution series of Jak Inhibitor 1. Cells were then fixed and the nucleus to cytoplasm translocation was measured using the Cellomics ArrayScan® V^TI  Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (3 µM Jak Inhibitor 1) and negative control (0.25% DMSO). The EC₅₀ of Jak Inhibitor 1 is approximately 40 nM in the assay.