Fig. 1

Comparison of migration, matrigel invasion and trans-endothelial migration ability of uPAR expressing A375 and uPAR lacking M14 melanoma cells. a Representative images of human A375 and M14 melanoma cells immune-stained with R4 anti-uPAR monoclonal antibody and visualized by a fluorescence inverted microscope. Nuclei were stained blue with DAPI. Scale bar: 5 μm. Original magnification: 1000 x. b Whole cell lysates (40 μg/sample) from A375 and M14 cells were resolved on a 10% SDS-PAGE followed by Western blotting with 1 μg/mL R4 anti-uPAR monoclonal antibody or 0.2 μg/mL anti-GAPDH polyclonal antibody as loading control. c Quantitative Real-Time PCR of uPAR in A375 and M14 melanoma cell lines. Results are the mean ± SD of three different experiments. **: p < 0.001. d-e Wound healing of A375 and M14 melanoma cells kept in growth medium at 37 °C, under a 5% CO₂ atmosphere. One field which includes the scratched path from each dish was selected and scanned sequentially every 30 min for 24 h. Images were recorded at the indicated times by a video-camera connected with a motorized inverted microscope. (Original magnification: 50×). e Square root of the wound area measured at the indicated times. f-g A375 and M14 cell migration (f) or matrigel invasion (g) toward serum-free medium (CTRL), or medium containing 10% FBS as source of chemoattractants, monitored for the indicated times by the xCELLigence system. Data represent mean ± SD from a quadruplicate experiment. h Trans-endothelial migration of A375 and M14 melanoma cells. HUVECs (1 × 10⁴ cells/well) suspended in growth medium, were allowed to grow for 24 h until they formed a confluent monolayer, prior to seeding melanoma cells (1 × 10⁴ cells/well). The breaking of monolayer integrity was monitored in real-time as changes in Cell Index for additional 5 h. Data represent mean ± SD from a quadruplicate experiment