2a, ?,b)

2a, ?,b).b). many tumors drive the manifestation of an additional set of pSTAT3-independent genes Rabbit Polyclonal to OR2D2 that contribute to tumorigenesis. In this chapter, we LY 541850 describe the HCS assay methods to measure IL-6-induced STAT3 signaling pathway activation in head and neck tumor cell lines as revealed by the expression and subcellular distribution of pSTAT3-Y705, pSTAT3-S727, and U-STAT3. Only the larger dynamic range provided by the pSTAT3-Y705 antibody would be robust and reproducible enough for screening. and MRAS) that do not respond to pSTAT3-Y705 [24C26]. U-STAT3 drives gene expression by a mechanism distinct from that used by pSTAT3-Y705 dimers [24C26]. U-STAT3 competes with IB for the un-phosphorylated NFB (p65/p50, U-NFB)) and the STAT3 NLS of the resulting U-STAT3::U-NFB complexes promotes their accumulation in the nucleus where they active a subset of B-responsive target genes [24C26]. The abnormally elevated levels of U-STAT3 produced by the constitutive activation of pSTAT3-Y705 observed in many tumors drive the expression of an additional set of pSTAT3-independent genes that contribute to tumorigenesis [24C26]. Head and neck cancer (HNC) is the eighth leading cause of cancer worldwide with a projected incidence of 540,000 new cases and 271,000 deaths per annum [27C29]. The front line therapies for HNC are surgical resection and chemo-radiotherapy. Surgical therapy can be disfiguring and there can be significant deleterious effects on swallowing, speech, and appearance. Radiation and chemotherapy treatment has produced limited improvement in prognosis and the 5-year survival rate for HNC has LY 541850 remained at 50% for over 30 years. There is, therefore, a need for new effective therapies. Elevated levels of activated STAT3 are frequently detected in HNC tumor samples and in head and neck squamous cell carcinoma cell lines used in mouse xenograft models that respond to STAT3 inhibition [4, 5, 7,10, 15, 30, 31]. STAT3 constitutively activated in an EGFR-independent manner by the autocrine/paracrine activation of the IL-6 receptor complex in HNSCC cells provides growth and survival benefits and may contribute to their resistance to EGFR-targeted therapies [16]. We recently described the development, optimization, and validation of high content imaging (HCS) assays to measure IL-6-induced pSTAT3-Y705 and interferon-gamma (IFN)-induced pSTAT1-Y701 levels in HNC cell lines [32], and used these phenotypic assays to screen for compounds that selectively inhibited STAT3 but not STAT1 path-way activation in HNSCC cells [33]. The IL-6-induced pSTAT3-Y705 and IFN-induced pSTAT1-Y701 HCS assays have subsequently been used to support the chemical lead optimization of hits that selectively inhibited STAT3 but not STAT1 pathway activation in HNSCC cells [33C35]. In this chapter, we describe the HCS assay method to measure IL-6-induced pSTAT3-Y705 activation, and because STAT3 S727 phosphorylation is required to achieve maximal STAT3 transcriptional activity and elevated levels of U-STAT3 drive the expression of genes that may LY 541850 contribute to tumorigenesis, we also describe methods to measure the expression and subcellular distribution of pSTAT3-S727 and U-STAT3. Cal33 HNSCC cells that had been seeded into 384-well plates in serum containing medium were cultured overnight and then serum starved for 24 h prior to a 15 min treatment 50 ng/mL IL-6. The cells were then fixed in 3.7% paraformaldehyde containing Hoechst, permeabilized with methanol, and then stained with the indicated primary anti-STAT3 antibodies and a FITC-conjugated anti-species secondary antibody. We then utilized the ImageXpress Ultra (IXU) confocal automated imaging platform to sequentially acquire images for the Hoechst (Ch 1) and FITC (Ch 2) fluorescent channels using a 20 0.45 NAELWD objective (Fig. 2). Fluorescent images of Hoechst stained nuclei from Cal33 cells acquired on the IXU were unaffected by treatment with IL-6 (Fig. 2a). In untreated Cal33 cells immuno-stained with a rabbit polyclonal antibody that recognizes total-STAT3 independently of its phosphorylation status, uniform STAT3 staining was observed throughout the cytoplasm and nuclear compartments in both gray-scale and color composite images (Fig. 2a, ?,b).b). In Cal33 cells that had been exposed to IL-6 however, there was an apparent increase in STAT3 staining in the nuclear compartment even though STAT3 was also still apparent in the cytoplasm (Fig. 2a, ?,b).b). In Cal33 cells immuno-stained with mouse monoclonal antibodies that recognize pSTAT3-Y705 or pSTAT3-S727, the indirect immuno-fluorescent staining was clearly higher in images acquired of Cal33 cells treated with IL-6 compared to those of un-stimulated cells (Fig. 2b). The color composite overlays of the Ch 1 and Ch 2 fluorescent images indicate that the increased immuno-fluorescent LY 541850 pSTAT3-Y705 and pSTAT3-S727 staining are predominantly localized within the nuclei of IL-6-treated Cal33 cells (Fig. 2b). Open in a separate window Fig. 2 Images of IL-6-induced STAT3 signaling pathway activation. (a) Grayscale and color composite images.