RI-3 prevents melanoma cell invasion through collagen I matrices contracted by dermal fibroblasts. a Whole cell lysates (40 μg/sample) of A375 or A375M6 melanoma cells were resolved on a 10% SDS-PAGE followed by Western blotting with 1 μg/mL R4 anti-uPAR monoclonal antibody, 1 μg/mL anti-FPR1 polyclonal antibody or 0.2 μg/mL anti-GAPDH polyclonal antibody, the last as loading controls. Lower panel: 50 μl of concentrated conditioned medium from A375 and A375M6 cells were resolved on a 10% SDS-PAGE under unreducing conditions followed by Western blotting with 1 μg/mL 389 anti-uPA polyclonal antibody. Bar graphs showing the average quantification of the uPAR/GAPDH and FPR1/GAPDH content from 3 independent experiments. Statistical significance with **p < 0.001.
b A375 and A375M6 melanoma cells were allowed to invade matrigel for 18 h in Boyden chambers toward serum-free medium (CTRL) or medium containing 10% FBS (FBS), in the absence (None) or the presence of 10 nM RI-3. The extent of cell invasion was expressed as a percentage of the A375 basal cell invasion 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 < 0.0001. c Trans-endothelial migration of A375 and A375M6 cells plus/minus 10 nM RI-3. Data represent mean ± SD from a quadruplicate experiment. d Photographs showing A375 and A375M6 cell invasion of collagen I matrices contracted by dermal fibroblasts in the absence or presence of 10 nM RI-3. Original magnification. 100×