Figure 4

Specific inhibition of MT1-MMP blocks HIF-2α mediated RCC tumor cell invasion. A. MT1-MMP protein expression as quantitated by an MT1-MMP ELISA activity assay. WT8 cells were transfected for 48 hours with pCMV-HIF-2α and a control or 3 specific MT1-MMP siRNA oligos. Each transfectant was additionally co-transfected with pCMV-eGFP. Transfection efficiency was consistent among the transfectants and was approximately 50%. Values represent the average [ng/mL] MT1-MMP of three transfections normalized to [μg/mL] total protein and are representative of three experiments (mean +/-S.D.); P < 0.005(**), P < 0.0005 (***) compared to the control siRNA. (B) and (C) WT8 cells were transfected for 48 hours with a control empty vector, pCMV-HIF-2α alone, or pCMV-HIF-2α and control or 3 specific MT1-MMP siRNA oligos. Each transfectant was additionally co-transfected with pCMV-eGFP. Transfection efficiency was approximately 50%. B. Western blot analysis of HIF-2α protein from whole cell lysates of the transfectants. Actin was used as a loading control. C. In vitro invasion assay of the transfectants using type I collagen coated FluoroBlok™ membrane inserts as described in Fig 3. Invaded cells were viewed by GFP fluorescence and counted from the entire membranes at 40×. Values represent the average number of invaded cells from three separate transfections set as relative invasion of the empty vector control and are representative of two experiments (mean+/-S.D.); P < 0.05 (#),P < 0.005 (##) compared to empty vector control; P < 0.0005 (***) compared to control siRNA. Statistical analyses were performed using the student's t-test.