Fig. 2
Relevance of uPAR to migration and trans-endothelial migration of melanoma cells. M14 cells were stably transfected with pcDNA3 empty vector (mock) or pcDNA3 loaded with full length uPAR (uPAR). a Whole cell lysates (40 μg/sample) from wild type (wt) or transfected M14 cells were resolved on a 10% SDS-PAGE followed by Western blotting with R4 anti-uPAR monoclonal antibody or anti-GAPDH polyclonal antibody as loading control. b Quantitative Real-Time PCR of uPAR in mock and in uPAR transfected M14 cells. Results are the mean ± SD of three different experiments. **: p < 0.001. c Cell migration of wild type (wt), mock and uPAR expressing M14 melanoma cells toward serum-free medium (CTRL), or medium containing 10% FBS monitored for 12 h by the xCELLigence system. Data represent mean ± SD from a quadruplicate experiment. d Trans-endothelial migration of wild type, mock and uPAR expressing M14 melanoma cells. Data represent mean ± SD from a quadruplicate experiment. e Whole cell lysates (40 μg/sample) from A375 cells transfected with siRNA targeting uPAR (uPARsiRNA), CTRLsiRNA or HyPerFect alone for 96 h were resolved on a 10% SDS-PAGE followed by Western blotting with R4 anti-uPAR monoclonal antibody or anti-GAPDH polyclonal antibody as loading control. The enclosed bar graph shows the average quantification of the uPAR/GAPDH content from 3 independent experiments. f Cell migration of A375 cells transfected with uPARsiRNA, CTRLsiRNA or HyPerFect for 96 h. Cells were allowed to migrate for 4 h at 37 °C in 5% CO2 in Boyden chambers toward serum-free medium (CTRL) or medium containing 10% FBS (FBS). The extent of cell migration was expressed as a percentage of the basal cell migration assessed in the absence of chemoattractant, considered as 100% (CTRL). Data are expressed as the mean ± SD of three independent experiments, performed in triplicate. Statistical significance with ** p < 0.001, ***p < 0.0001. g Trans-endothelial migration of A375 cells transfected with uPARsiRNA, CTRLsiRNA or HyPerFect alone. Data represent mean ± SD from a quadruplicate experiment