Figure 6

Okadaic acid (OA) reduced PRV replication in PRV-infected PK15 cells. A PK15 cells were infected with PRV (MOI = 0.1) in cell culture medium containing 1 µM Tg and were then harvested at 24 hpi. Western blot analysis was performed to measure p-PERK, PERK, p-eIF2α, and eIF2α protein levels. B PK15 cells were infected with PRV (MOI = 0.1) and incubated at 37 °C for 8 h before the addition of OA at the indicated concentrations. Cells were then incubated for another 15 h in the presence of OA, followed by puromycin labelling for 1 h. Cells were harvested at 24 hpi. Western blot analysis was performed to measure p-eIF2α, eIF2α, and PRV protein levels. The intensities of the p-eIF2α bands were determined by densitometry, normalized to eIF2α, and shown as fold changes (bottom panel). The values are presented as the mean ± SD of triplicate experiments. ** p < 0.01; *** p < 0.001. C De novo protein synthesis and PRV protein expression were measured by Western blot analysis; the intensities of bands corresponding to puromycin-labelled proteins were quantified by densitometry and normalized to β-actin and are shown as fold changes. The intensities of the PRV protein bands were quantified by densitometry and normalized to β-actin and are shown as fold changes (right panels). The values are presented as the mean ± SD of triplicate experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. D, E PK15 cells were infected with PRV (MOI = 0.1) and incubated at 37 °C for 8 h before the addition of OA at the indicated concentrations. Cells were then incubated for another 16 h. Supernatants and cells were harvested at 24 hpi. RT–qPCR was performed to determine the relative mRNA level of PRV-gE compared to β-actin (D); the viral titre in the supernatants was determined in PK15 cells (E). The data are presented as the mean ± SD of three independent experiments. ** p < 0.01; *** p < 0.001.