Figure 8

Knockdown of GADD34 in PK15 cells inhibited PRV replication. A PK15 cells were transfected with shNC or shGADD34 (#1, #2, #3, and #4) for 24 h and were then infected with PRV (MOI = 0.1). Cells were subjected to puromycin labelling for 1 h at 23 hpi and were then harvested at 24 hpi. Western blot analysis was performed to detect p-eIF2α, eIF2α, and PRV proteins; GADD34 and PRV protein expression was quantified by densitometry and normalized to β-actin and are shown as fold changes. The intensities of the p-eIF2α bands were quantified by densitometry, normalized to eIF2α, and shown as fold changes (bottom panels). The values are presented as the mean ± SD of triplicate experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. B De novo protein synthesis was measured in GADD34 knockdown cells by using a monoclonal antibody against puromycin; 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 lower panel). The values are presented as the mean ± SD of triplicate experiments. ** p < 0.01. C, D PK15 cells were transfected with shNC or shGADD34 (#1, #2, #3, and #4) for 24 h and were then infected with PRV (MOI = 0.1). 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 (C); the viral titre in the supernatants was determined in PK15 cells (D). The data are presented as the mean ± SD of three independent experiments. *** p < 0.001.