Video camera

Cells (~ 3 × 10⁴/sample) were seeded on glass coverslips and cultured for 24 h in growth medium. Then, slides were washed with PBS, fixed with 2.5% formaldehyde in PBS for 10 min at 4 °C and then incubated with 2 μg/mL anti-R4 anti-uPAR monoclonal antibody recognizing uPAR D3 domain (1 μg/mL) [18 (link)], 2 μg/mL rabbit anti-αV polyclonal antibody from Abcam (EPR16800) for 2 h at RT, permeabilized with 0.1% Triton X-100 in PBS for 10 min at 4 °C and then incubated for additional 2 h at RT with 2 μg/mL guinea pig anti-cytokeratin 18 polyclonal antibody (Progen) (Fig. 3a). Anti-αV polyclonal antibody from Abcam, permeabilization step and guinea pig anti-cytokeratin 18 (CK18) were employed for Fig. 3b. Then, after three washes with PBS, 1:800 Alexa Fluor 488-conjugated F(ab’)2 fragment of rabbit anti-rabbit IgG, or Alexa Fluor 594 goat anti-guinea pig IgG antibodies, both purchased from Molecular Probes were applied to slides at RT for 45 min. After nuclear staining with 4–6-diamidino-2-phenylindole dye (DAPI), coverslips were mounted using 20% (w/v) mowiol and analyzed by a fluorescence inverted microscope connected to a video-camera (Carl Zeiss) or by the LMS510 Zeiss confocal microscope (Carl Zeiss).

To analyze cytoskeletal organization, cells grown on glass slides to semi-confluence, were fixed with 2.5% formaldehyde, permeabilized with 0.1% Triton X-100 for 10 minutes at 4°C, and then incubated with 0.1 μg/ml rhodamine-conjugated phalloidin (Sigma-Aldrich) for 40 minutes. After nuclear staining with 4-6-diamidino-2-phenylindole dye (DAPI), cells were analysed by a fluorescence inverted microscope connected to a videocamera (Carl Zeiss).

Organotypic culture system was carried out as described by Timpson and coworkers [48 ]. Briefly, 1 × 10⁵ NHDF normal, dermal fibroblasts were starved in serum-free medium for 18 h, suspended in 250 μl FBS and embedded in 250 μl alpha Minimum Essential Medium 10× (αMEM 10×) containing 2 mg/mL Type I Collagen (#124–25; Cell Application INC.). Collagen/fibroblast mixture (2.5 ml/well) was plated in 35 mm plastic dishes and allowed to polymerize for 1 h at 37 °C, before the addition of 2 mL growth medium. Collagen/fibroblast matrix was allowed to contract until it fitted in a 24-well dish (~8 days), changing media every other day. Then, 1 × 10⁵ melanoma cells were seeded on top of the matrix and allowed to grow for 72 h, before transferring the matrix to a grid (Screens for CD-1™ size 40 mesh S0770 Sigma) in order to create an air/liquid interface and a chemotactic gradient that promotes cell invasion. Melanoma cells were allowed to invade replacing growth medium, with/without 10 nM RI3, every 2 days. After 14 days, matrixes were cut in half, fixed with 10% formalin and processed for paraffin embedding. Microtome sections about 5 μm thick, were stained with hematoxylin and eosin solutions and analyzed by using a microscope connected to a video camera (Carl Zeiss).