For tyrosinase: annealing at 52°C for 30

s, extension at 73°C for 60 s and denaturation at 95°C for 45 s and a final cycle with a 5 min long extension. For E5 the E5P65 sense (TGC ATC CAC AAC ATT ACT GGC G) and E5M3AS antisense (AAC ACC TAA ACG CAG AGG CTG C) primers were used; for human tyrosinase the primers were Hu-TYR1 (TTG GCA GAT TGT CTG TAG CC) and Hu-TYR2 (AGG CAT TGT GCA TGC TGC TT) as suggested by Calogero et al. [32]. Cell viability, cell proliferation and cell specific metabolic activity Cell viability was measured as already described [27], Briefly, cells AZD5582 mw were seeded in 96-well microplates at a density which allowed an exponential growth rate for the following 5 day incubation (i.e. 1.0 × 104/well for M14 and 1.6 × 104/well for FRM). At 24 h intervals the cells were challenged with 1.25 mg/ml MTT in a 100 μl volume of fresh medium containing 0.1% FBS [33]. After 2 h of incubation the monolayers were then decanted, washed twice with PBS and the reduced insoluble dye eluted

by 100 μl of isopropanol/HCl 0.04 N. The cell viability was then assessed through the MTT reducing activity evaluated by the A540 – A750 difference measured by a microplate reader (Labsystem Multiscan MS – Thermo Fisher Scientific, Inc. Waltham MA). Cell proliferation was measured by the growth curve as already described [34]. Briefly, cells were seeded in 96-well microplates at the same density as above. At 24 h intervals the monolayers were Nutlin-3a molecular weight stained with Crystal

Violet (CV), the dye was eluted by means of 33% acetic acid and the cell number in each well was estimated by the A540 measured in a microplate reader (Labsystem). Considering that cell viability assay does actually measure the total reducing activity within a tissue culture, and considering that such a global activity may largely vary according to culture conditions, cell environment and phenotypic Thiamet G status, to gain information about a possible modulation of the metabolic activity within E5 expressing cells, the cell specific metabolic activity was calculated. This is the simple MTT/CV absorbance ratio, expressed in arbitrary units, and gives information about the average metabolic activity of single cells. For each assay a set of at least four different experiment was considered. Each experiment consisted of eight independent replicas. Acridine orange fluorescent staining To visualize acidic organelles, Acridine orange (AO) was used [35]. AO is a fluorescent probe that emits green at low concentration and orange at high concentration. To determine the effect of treatments on endocellular compartment pH, cell cultures were seeded onto multiwell microscope slides and allowed to attach overnight. The culture medium was then replaced with non supplemented medium or medium containing 10 nM ConA or medium containing the retrovirus.