Expression of VHL inhibits collagen invasion and degradation. A. Collagen invasion. WT8 and pRc-9 cells were cultured in serum-free media on top of type I collagen coated membranes in a Transwell^® invasion assay system. The lower chambers contained either serum-free DMEM as a negative control or DMEM supplemented with 10% FBS as a chemoattractant. At time of harvest, the non-invaded cells and collagen matrix on top of the membranes were removed, and invaded cells on the bottoms of the membranes were stained with methylene blue. A representative picture is shown of cells invading towards serum-free versus serum-containing media. Cells were counted from 3 fields per sample at 100× and averaged. Values in the graph represent the average number of cells invaded towards serum from four separate experiments relative to WT8 cell invasion (mean+/-S.D.); P < 0.05 (*). B. Collagen degradation. WT8 and pRc-9 cells were serum-starved overnight, harvested and then embedded in a mixture of type I collagen and serum-free DMEM for the collagen degradation assay. The collagen gel was allowed to solidify and serum-free media was added to the top of the collagen gel. The overlying media was weighed 48 hours after time of plating, and collagen degradation was determined by the volume of media liberated from the collagen gel. Values represent the average μL of media released from three samples (mean+/-S.D.); P < 0.01 (*) and are representative of four separate experiments. Statistical analyses were performed using the student's t-test.

Expression of HIF-2α or MT1-MMP in WT8 cells increases matrix degradation and invasion. A. Collagen degradation. WT8 cells were transfected with either a control empty vector or pCMV-HIF-2α. Transfectants were serum-starved overnight, harvested and then embedded in a mixture of type I collagen and serum-free DMEM for the collagen degradation assay as in Fig 1. After 48 hours, the overlying media was weighed, and collagen degradation was determined by the volume of media liberated from the collagen gel. Values represent the average μL of media released from six samples (mean+/-S.D.); P < 0.0001 (***). B. Quantitative real-time RT-PCR analysis of MT1-MMP mRNA expression in WT8 cells co-transfected with pCMV-eGFP and either a control empty vector or MTpc3SE. Values represent the average pg of MT1-MMP mRNA normalized to ng of GFP mRNA from three separate transfections (mean+/-S.D.); P < 0.0001 (***). C. Collagen degradation. WT8 cells were transfected with either a control empty vector or MTpc3SE. Transfectants were subjected to the collagen degradation assay as in Fig 1. After 48 hours, the overlying media was weighed, and collagen degradation was determined by the volume of media liberated from the collagen gel. Values represent the average μL of media released from six samples (mean+/-S.D.); P < 0.0001 (***). D. Collagen invasion. WT8 cells were transfected with either an empty vector or MTpc3SE. Transfectants were subjected to a collagen invasion assay as described for Fig 1. At time of harvest, the non-invaded cells and collagen matrix on top of the membranes were removed and membranes stained with methylene blue. Cells were counted from 3 fields per sample at 100× and averaged. Values represent the average number of invaded cells from five separate experiments set as relative cell invasion of the empty vector transfectant (mean+/-S.D.); P < 0.005 (**). Statistical analyses were performed using the student's t-test.

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.

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.