Chitosan-Based Delivery of Avian Reovirus Fusogenic Protein p10 Gene: In Vitro and In Vivo Studies towards a New Vaccine against Melanoma

Figure 2

ARV-p10 protein expression in B16 melanoma cells using NP-ARV. (a) The expression of the ARV-p10 protein (upper panel) and housekeeping GAPDH (lower panel) transcripts was determined by RT-PCR 48 hours posttransfection. A representative gel containing DNA ladder (L, lane 1), PCR blank control (control, lane 2), nontransfected cells (parental, lane 3), Lipofectamine pIRES-ARV-transfected cells (Lipo-ARV, lane 4), and NP-ARV-transfected cells (lane 5). (b) Expression of ARV-p10 protein determined by immunofluorescence. B16 (upper panel), Lipo-ARV-transfected B16 cells (middle panel), and NP-ARV-transfected B16 cells (lower panel) stained with DAPI (left column) and with an antibody against ARV-p10 protein (middle column). Merge is shown in the right column. (c) Syncytium formation 48 h posttransfection of B16 cells (upper panel), Lipo-ARV-transfected B16 cells (middle panel), and NP-ARV-transfected B16 cells (lower panel) stained with DAPI (left column) and CellMask (middle column). Merge is shown in the right column. White arrows mark fusion points. (d) Quantification of syncytia 48 hours posttransfection by hemacolor stain. (e) Cell viability was evaluated at 24, 48, and 120 hours posttransfection and was normalized against nontransfected cells. Graphs correspond to of three independent experiments. Statistical analyses were performed using the Mann-Whitney test ().

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