Contact Induced Regulatory DC Figure 3. JSI-124 inhibits IL-10 production induced by cell:cell contact but not by tumor cell’s conditioned media. 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 (PC3 or MCF-7, as indicated; lower panels) or with tumor cell’s conditioned media (upper panel). After 24 hours, cells were harvested and IL-10 production was detected by intracellular immunostaining and flow cytometric analysis. Monocytes that were gated based on their forward- and side-scatter characteristics are shown. Dashed line: Isotype control; Filled grey area: monocytes alone; Thick grey line: monocytes that were either co-cultured with tumor cells (lower panels) or were cultured in MCF-7 conditioned media (upper panel); Black line: JSI-124-treated monocytes incubated with tumor cells or their conditioned media, as above. Similar results were obtained in three independent experiments. doi:10.1371/journal.pone.0006846.g003 treatment was further demonstrated using anti-phosphorylated STAT3 immunoblotting using both monocytes and DC co- cultured with MCF-7 cells (Fig. 2C). Hence, using these specific conditions (i.e. 1 h pre-treatment prior to stimulation) we could discriminate between the soluble factor- and contact- dependent STAT3 activating pathways. These results further support that in tumor-APC interaction there is a contact-dependent activation of STAT3 in APC, that unlike the parallel cytokine-mediated pathway, is sensitive to JSI-124 inhibition (as is the case with hMSC-APC co-cultures [13]). Cell:cell contact but not conditioned media or IL-10 induce JAK2 phosphorylation To further demonstrate the involvement of JAK2 in contact- dependent STAT3 activation, we directly assessed JAK2 phosphor- ylation in cell extracts from the various treatments using SDS-PAGE and immunoblotting using anti-phosphotyrosine JAK2. A band corresponding to phosphorylated JAK2 appeared only when monocytes and tumor cells (MCF-7) were co-cultured together (Fig. 5A) but not when either conditioned media or IL-10, were used (Fig. 5B and C, respectively). As expected, this contact-dependent JAK2 phosphorylation was significantly reduced upon pretreatment of monocytes with JSI-124, paralleling that of phosphorylated STAT3 (Fig. 5A). Significantly, while no phosphorylated JAK2 was detected in either conditioned media or IL-10 treated monocytes, phosphorylated STAT3 was readily detected and was insensitive to JSI-124 treatment, (Fig. 5B and C and 6B). Of note, since only monocytes were pre-treated with the inhibitor JSI-124, the reduced level of phosphorylation of both JAK2 and STAT3 in the co-cultures strongly suggests that these events originate in the monocytes. phosphorylated-STAT3 co-immunoprecipitates with JAK2 upon MCF7:monocyte cell contact but not IL-10 treatment Furthermore, we found that JAK2 and STAT3 are physically associated and that the latter was tyrosine-phosphorylated after PLoS ONE | www.plosone.org 4 August 2009 | Volume 4 | Issue 8 | e6846