Figure 4

Functional Role of SOX1 during invasion. A) The Trans-Lentiviral pTRIPZ system from Open Biosystems was used to introduce shRNA against BMX, SOX1 or a non-silencing control vector in DU145 cells. The cells were selected for 2 weeks in 1 μg/mL of puromycin and single cell clones were generated. To induce expression of the shRNA 1 μg/mL of doxycycline was added. The plasmid is designed to have a TET inducible TurboRFP upstream of the shRNA and they should appear red upon successful infection. B) Lowered expression was confirmed using Western blotting. Follow up experiments were conducted using BMX clone 3 and 5 and SOX1 clone 7 and 8 since they demonstrated the most significant decrease in protein expression. Fold changes represent samples normalized to actin and the control level of expression. C) Proliferation assays were conduced using Cell Titer-Glo kit and assayed on Day 1, 3, 5 and 7. More proliferation is indicated by an increase in relative luciferase units (RLUs). *denotes statistical significant p < 0.05 compared to vector transfected cells. A significant decrease was observed in shSOX1 #7 cells compared to vector transfected cells, and a significant increase was observed in shBMX #5 cell line. D) Matrigel invasion assays were conducted for 24 hours toward SCM. Top cells were removed and bottom cells were stained with the Diff-Quick staining kit from Dade Behring. Cells were counted using 4 independent fields per sample and 2 chambers were used per cell line. *denotes statistical significance p < 0.05 compared to vector transfected cells. Both shSOX #7 and #8 demonstrated significant decreases in invasion toward SCM compared to vector transfected cells.