Lane 1: DNA from cells infected with the control retrovirus; Lane 2: DNA from cells infected with the HPV-16 E5 retrovirus; Lane 3: DNA digested total RNA from cells infected with the HPV-16 E5 retrovirus; Lane 4: Non retrotrascribed DNA digested total RNA from cells infected with the HPV-16 E5 retrovirus; Lane 5: No template negative control; Lane 6 positive control (0.5 μg Siha cell DNA). MW: DNA molecular buy CFTRinh-172 weight marker VIII (Roche Selleckchem Idasanutlin Biochemicals SpA): arrows on the left-hand side indicate the bp length of some reference bands. The band with size of 160 bp (left sided empty arrow) demonstrate the presence of viral

E5 sequence and its transcription. Four independent experiments gave similar results. Figure 2 Effect of HPV-16 E5 expression on the proliferation, cell viability and on cell specific metabolic

activity of M14 and FRM melanoma cells. Cell proliferation (upper row) was slightly decreased in E5 expressing cells (empty symbols) as compared with control cells (full symbols). The cell viability of E5 expressing cells and control cells is shown in the middle row. The cell specific activity of E5 expressing cells (lower row) was higher than that of control cells. This effect, sharply evident in FRM cells appeared slighter in M14 and indicates an increased oxidative metabolism in E5 expressing cells. Values are the mean ± S.D. of eight independent replicas and are derived from a representative experiment in a set of four. Statistical

comparison of E5 expressing cells was made using either parametric BAY 63-2521 mw (Student’s t -test) or non paramentric (Mann – Whitney test) according to the results of the Shapiro – Wilk assay. (* = p < 0.05; ** = p < 0.005). The specific metabolic activities are calculated as the simple cell viability/cell proliferation ratio (MTT/CV ratio) and are expressed in arbitrary units as the mean of four different experiments ± SD. E5 expression modulates endosomal pH and restores tyrosinase activity Being well accepted the biochemical interaction of E5 with the V-ATPase proton pump, we investigated Dichloromethane dehalogenase if the infection with E5 could determine pH changes in FRM and M14 cells. The fluorescent stain Acridine Orange (AO) used for analysis is an acidotropic weak base which is taken up by living cells and accumulates in acidified compartments such as lysosomes, and melanosomes. When AO accumulates at high concentrations in acidic environment the fluorescence is orange; while at low concentration AO emits green [33]. The effect of E5 expression on endosomal pH is shown in Fig. 3. In E5 expressing cells (+E5), the replacement of orange fluorescence with green fluorescence indicated the raise of intracellular pH with respect to control cells. The addition of the proton pump inhibitor Con-A, a recognised alkalinizing agent, to control cells determined a similar colour change of fluorescence indicating that alkalinisation occurred.