Direct interaction between SOX1 and STAT3. A) Matrigel invasion assays were performed for 24 hours toward SCM using DU145 cells in the presence of the anti-IL-6, the PI3K inhibitor LY294002, a small molecular inhibitor of MEK called U0126 (thus downstream inhibition of extracellular-related kinase (ERK1 and ERK2) mediated responses), a small molecule inhibitor JAK called AG490 (Janus Kinase) and an inhibitor of its partner signal transducers and activators of transcription-3 (STAT3) called Static or the Tec kinase family inhibitor LFM-A13.Significant differences were observed between control cells and those cells treated with U0126, Stattic, LY294002 and LFM-A13. B) qRT-PCR analysis was performed as mentioned in Figure 3. *denotes statistical significant p < 0.05 compared to the non-invasive cells. Increased levels of Stat3 are seen invasive LNCaP and DU145 cells compared to the parental lines. C) Staining of pSTAT3 in invasive or non-invasive DU145 cells was performed directly on the Matrigel membrane and carried out as previously described in Figure 3. D) DU145 lysates were incubated with either SOX1, STAT3 or BMX overnight at 4°C with rotation. Samples were then incubated with Protein A-agarose beads to isolate complexes. Membranes were then incubated overnight at 4°C using primary antibodies for STAT3 or SOX1. The membrane was developed using the Odyssey from Licor. Protein loading was normalized using actin as a control. E) Western blotting for STAT3 and pSTAT3 in sub-cellular protein extracts from DU145, NS, shBMX#3 or shSOX1#8. F) STAT3 EMSA: Each lane contains WT-IR STAT3 oligos. Lane 1-3 DU145, 4 and 5 NS, 6 and 7 shBMX#3, 8 and 9 shSOX1 #8 and 10 contains no protein. Lane 2 contains excess MU-IR STAT3 and lanes 3, 5, 7 and 9 contain excess unlabeled WT probe. Supershifited samples appear below and only contain WT-IR STAT3.