H-1PV infection of MZ7-Mel cells. A: Effect of infection on cell survival. MZ7-Mel cells were infected with H-1PV (MOI = 20 PFU/cell), grown for 4 to 6 days and stained with crystal violet. The percentage of survival is expressed as fraction of dead versus living cells by photometric analysis (mean of 24 wells, at least 2 experiments). B: NS1 gene expression. MZ7-Mel cells were infected and grown for 1 to 4 days. After lysis 50 μg of total proteins were equally diluted and separated on SDS-PAGE. For parvoviral protein detection, blots were incubated with the NS1-specific antibody. C: Virus-driven transgene expression. Cells were infected with recombinant hH1Δ1600luc (MOI = 10-2 RU/cell) and transgene activities were measured in tumor cell lysates after 3 and 4 days p.i., given in fold induction. D: Phagocytosis. Immature DC and MZ7-Mel cells were labeled with PKH2 and PKH26, respectively. MZ7-Mel cells were infected with H-1PV (MOI = 20 PFU/cell), cultured for 10 days, and subsequently co-cultured with iDC. After 2 days of co-culture, PKH2-labeled DC were analyzed by FACS for their uptake of PKH26-stained MZ7-Mel cell lysates. As controls, DC were incubated with melanoma cells that were either untreated, UV-irradiated (to induce apoptosis) or ultrasonic-treated (necrosis). The percentage indicates the proportion of double-positive (phagocytosing) cells. FL-1 corresponds to an area in which PKH2 and PKH26 fluorescence overlap. The dotted curve represents the untreated DC. E: DC maturation. The expression of CD80, CD83, and CD86 was measured by FACScan after immature DC were incubated for 2 days with untreated SK29 melanoma cells, with UV-irradiated cells, and lysates from H-1PV-infected cells (MOI = 20 PFU/cell) 10 days p.i.