Contact Induced Regulatory DC Figure 2. Contact-dependent but not conditioned media-induced STAT3 activity is sensitive to JSI-124 inhibition. A. Monocytes were either left untreated or treated with JSI-124 (10 mM) for 1 hour at 37uC, and then washed. Treated and untreated cells were then cultured alone or were co-cultured with either MCF-7 or PC3 cells (upper panels) or with their conditioned media (lower panels). After 2 hours, nuclear extracts were prepared and tested for STAT3 activity using Non-Radioactive STAT3 Transcription Factor Assay. Comparable results were obtained in four separate experiments. B. Monocytes were either left untreated or treated with JSI-124 (10 mM) for 1 hour at 37uC, and then washed. JSI-124-treated and untreated cells were then cultured alone or co-cultured together with tumor cells (MCF-7) or with tumor cell’s conditioned media. After 2 hours, cells were harvested and phosphorylated-STAT3 was detected by intracellular immunostaining and flow cytometric analysis. The two cell types were distinguished based on their forward and side scatter characteristics and phosphorylated STAT3 staining in monocyte population is shown. Dashed line: isotype control; Filled grey area: monocytes alone; Thick grey line: monocytes that were either co-cultured with MCF-7 (left panel) or were cultured in MCF-7 conditioned media (right panel); Black line: JSI-124-treated monocytes incubated with MCF-7 or their conditioned media, as above. Comparable results were obtained in three independent experiments. C. Monocytes or DCs were either left untreated or treated with JSI-124 (as indicated above), and then were co-cultured with MCF-7, for 2 hours. Untreated cells were obtained by mixing MCF-7 and either monocytes or DC without incubation. Cell extracts were subjected to SDS-PAGE and immunoblotting of anti-phosphorylated STAT3 demonstrate induction of STAT3 and that JSI-124 treatment blocks contact-dependent STAT3 phosphorylation. Anti-STAT3 immunoblotting reveals relative amounts of protein in each lane (lower panels). doi:10.1371/journal.pone.0006846.g002 individually, measured 2 hours later. Suggesting that cell-cell contact may also contribute to STAT3 induction in addition to soluble factors (Fig. 1). Furthermore, intracellular staining for phosphorylated STAT3 revealed that this contact-dependent increase resulted from STAT3 activity within the monocytes (Fig. 2B). Contact-dependent but not conditioned media or IL-10- induced STAT3 activity and IL-10 production is sensitive to JSI-124 inhibition Cytokine receptors that lack an intrinsic tyrosine kinase activity such as receptors for IL-10 and IL-6 rely on JAKs for initiating their signaling cascade and STAT3 activation (reviewed in [4]). Our previous data on hMSC:APC co-cultures [13] demonstrated that STAT3 activity is abrogated upon treatment with JSI-124 (cucurbitacin), a small natural molecule that inhibits JAK2 signaling [15], suggesting that the contact dependent mechanism may involve JAK2 signaling pathways. To further test this, we have pretreated monocytes with JSI-124, for 1 hour prior to their co-culturing with the tumor cells, or incubation with either IL-10 or tumor cell’s conditioned media. Pretreatment of the cells was necessary in order to avoid JAK2/STAT3 inhibition in the tumor cells while focusing on signaling events within the APC. Pretreatment of monocytes with JSI-124, resulted in a significant reduction in STAT3 activity (measured by both STAT3 transcription factor assay (Fig. 2A, upper panels) or phospho- STAT3 intracellular immunostaining (Fig. 2B, left panel)) and in IL-10 production by the monocytes (Fig. 3, lower panels) in tumor cell:monocyte co-cultures. On the other hand, JSI-124 pre- treatment had no effect on STAT3 activity (Fig. 2A, lower panels), phospho-STAT3 immunostaining (Fig. 2B, right panel) or IL-10 production (Fig. 3, upper panel) when monocytes were incubated with MCF-7’s conditioned media. Likewise, JSI-124 had no effect on STAT3 activity (Fig. 4A) or phospho-STAT3 intracellular immunostaining (Fig. 4B) in IL-10-treated monocytes. This activation of STAT3 as well as its sensitivity to JSI-124 PLoS ONE | www.plosone.org 3 August 2009 | Volume 4 | Issue 8 | e6846