Figure 3

Inhibition of MT1-MMP blocks RCC tumor cell invasion. (A) and (B) WT8 cells were transfected for 48 hours with MTpc3SE and 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%. A. MT1-MMP protein expression in the transfectants as quantitated by an MT1-MMP ELISA activity assay. 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 (**) compared to control siRNA. B. Gelatin zymography of conditioned media from transfectants to measure the presence of active MMP-2. Cells were transfected overnight and then cultured in serum-free media to condition the media for 24 hours. Conditioned media was concentrated before analysis of gelatinolytic activity. C. In vitro invasion assay using type I collagen coated Fluoroblok™ membrane inserts. WT8 cells were transfected for 48 hours with a control empty vector, MTpc3SE alone, or MTpc3SE and control or 3 specific MT1-MMP siRNA oligos. Each transfectant was additionally co-transfected with pCMV-eGFP and transfection efficiency was approximately 50%. Transfectants were serum-starved overnight before culturing on top of the collagen coated membranes in serum-free media. The lower chambers contained DMEM supplemented with 10% FBS as a chemoattractant. 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 < 0.005 (##) compared to empty vector control; P < 0.0005 (***) compared to control siRNA. Statistical analyses were performed using the student's t-test.