Virus infection and transduction efficiency.

<p><b>A</b>) <i>Cytopathic effect of wtPV-H1</i>. The tumor cell lines BxPc3, Panc1 and AsPc1 were infected at MOIs ranging from 1 to 100 and after 72 hours, cell viability was measured by MTT assay. Indicated data represent the mean of three independent experiments realized in triplicate. <b>B</b>) For pancreatic tumor cell infection study, the cell cultures were blocked in G0/G1 phase by serum starvation for 72 hours and thereafter, tumor cells were infected with wtPV-H1or rPVH1-yCD at MOI 10 for 12 hours, and subsequently transferred on nitrocellulose filters. The detection of NS1 viral gene was realized according to the protocol of PV-H1 titration mentioned above. The detected radioactive spots reflect the number of NS1-hybridized probes. Asterisks indicate significant difference (*** p<0.001) observed between permissive cells (Panc1 and AsPc1) and non-permissive (or low) BxPc3 cells. <b>C</b>) Representative microscopy fluorescence images of AsPc1, Panc1 and BxPc3 cell cultures infected with rPVH1-GFP. Cells were infected at MOI 5 and 10 during 48 hours. <b>D</b>) Fluorescence intensity was quantified by flow cytometry. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070594#s3" target="_blank">Results</a> represent the percentage of fluorescent cells reported to total cells. <b>E</b>) For transduction efficiency. Kinetics of NS1 and yCD expression were carried out on AsPc1, Panc1 and BxPc3 cells. Tumor cells were infected with rPVH1-yCD (MOI 10) and total RNA extractions were performed 12, 24, 48 and 72 h post-infection and subjected to real time RT-PCR measurement. Relative quantitation of gene expression was achieved by normalization against the endogenous GAPDH housekeeping gene expression. Error bars represent the standard error of three independent experiments and run in quadruplets.