Figure 2

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.