The slides were fixed with 2% formaldehyde in PBS and processed

for fluorescence microscopy with a Zeiss 466301 microscope. An Olympus Camedia C5060 was used for colour photography. Anchorage independent growth assay A 2 ml of 0.5% agarose gel in RPMI at 10% FCS was poured in each 35 mm well of a plastic plate and allowed to solidify at room temperature for 2 hours in a laminar flow hood. Then a 0.5 ml of a 0.33% agarose gel containing 250 cells was overlaid on top, allowed to stand for 30′ at +4°C and subsequently incubated at 37°C. After a 12–16 days incubation the cell growth was evaluated by bright field FDA approved Drug Library observation under low magnification and growing colonies photographed. Western blot analysis Immunoblot analysis was performed as previously described [36]. Cell lysis was carried out at 4°C by sonication for 1 min in Media I (0.32 M sucrose, 10 mM Tris-HCl, pH 8.0, 0.1 mM MgCl2, 0.1 mM EDTA, 1 mM phenyl-methyl-sulfonyl-fluoride (PMSF) and 10 μg/ml aprotinine) and lysates were stored at -70°C until use. Protein

content was determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories Srl, click here Segrate, Italy). Proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes in 25 mM Tris, 92 mM glycine containing 20% (v/v) methanol at 110 V for 1 h. Following transfer, membranes were placed for 1 h in blocking buffer (bovine serum albumin 3% in T-TBS). For tyrosinase detection, membranes were probed first with 10 ml of blocking buffer containing goat anti-tyrosinase polyclonal antibody (Santa Cruz Biotechnology Inc., CA) (1:500) for Teicoplanin 1 h at 27°C, followed by 10 ml of blocking buffer containing horseradish peroxidase-conjugated rabbit anti-goat IgG (1:5000) for 60 min at 27°C. Protein bands were visualized using luminol-based enhanced chemo-luminescence as described by the manufacturer (Perkin-Elmer

Life Sciences). Densitometric analysis was performed using Scion Image (PC version of Macintosh-compatible NIH Image). Tyrosinase activity assay Cell monolayers were treated with trypsin/EDTA; suspensions washed with PBS and pellets recovered by centrifugation at 250 × g for 10 min. Cells were lysed by sonication (six times for 5 seconds each) in 0.5 ml of 0.1 M Na-phosphate buffer, pH 6.8, containing 0.1 mM PMSF. After centrifugation at 7,000 × g for 10 min, tyrosinase activity was assayed on supernatant according to Iozumi et al. [37]. Fifty μl of sample was incubated in 0.5 ml of a reaction mixture containing 0.1 mM L-tyrosine, 2 μCi per ml of [3H] tyrosine, 0.1 mM L-DOPA and 0.1 mM PMSF in sodium phosphate buffer 0.1 M (pH 6.8). After 2 h at 37°C, the reaction was terminated by the addition of 1 ml of charcoal (10% wt/vol in 0.1 N HCl). Samples were centrifuged at 2000 g for 10 min, the supernatant was removed and mixed with scintillation cocktail, and radioactivity was determined using the LS 6500 scintillation system (Beckman, U.S.A.).