Figure 4. ETS1 activates MMP-9 expression in presence of GSK3β. (A) The MMP-9 promoter-luciferase construct was co-transfected with a control vector (labeled as “-” at the bottom of the figure) and Flag-ETS1 in presence or absence of the GSK3β si in SKOV3, MDAH2774 and ARK2cells. (B) MMP-9 mRNA was detected with qPCR in overexpressing Flag-ETS1 or control vector cells in presence or absence of the siGSK3β. The MMP-9 promoter activity (A) and qPCR results (B) in cells transfected with the control vector (Flag-control and si-control) cells were set at 1. Results shown are the mean ± standard errors of the mean from three independent experiments. (C) Induction of MMP-9 by Flag-ETS1 (left panel) or Flag-ETS1 3A (right panel), was suppressed in cells in which the GSK3β expression was knocked-down for 72 h, and subsequently treated with MG132 (10 μM) for 5 h. The red arrows indicate the Flag-ETS1, Flag-ETS13A and mature MMP9. (D) ETS1 and MMP-9 expression in tumors from mice treated with vehicle or LY2090314 (10 mg/kg) twice a week for four weeks. Protein levels of ETS1, MMP-9, and actin were determined by western blot. Asterisks denote statistical significance (*p < 0.05, **p < 0.01).