Generation and characterization of ST-expressing B16/ST tumor cell line. B16 mouse melanoma cells were transduced with a lentiviral vector containing a mammalian codon-optimized gene encoding Merkel cell polyomavirus (strain 350) small T antigen (ST) under the control of cytomegalovirus and GFP reporter under EF1 promoter to generate tumorigenic B16/ST tumor cell line. (A) Characterization of the transduction of B16/ST tumor cells by flow cytometry analysis after sorting. B16/ST tumor cells (green) or control B16 melanoma cells (purple) were sorted and characterized for GFP expression by flow cytometry analysis. (B) Schematic diagram of vaccination schedule for Western Blot analysis. C57BL/6 mice were vaccinated intramuscularly by electroporation three times at 1-week intervals and boosted at the same dose. Western Blot analysis using sera from vaccinated mice was performed 1 month after last vaccination to determine ST protein levels of B16/ST cells. (C) ST protein levels determined by Western blot analysis. Membranes were probed with either serum from mice vaccinated with pcDNA3-MCC/ST or anti-β-actin antibody for loading control. Lane 1, negative control B16. Lane 2, B16-MCC/ST. (D) ST RNA levels determined by RT-PCR. Lane 1, negative control B16. Lane 2, B16-MCC/ST.