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. 2017 Dec 13;12(12):e0189191. doi: 10.1371/journal.pone.0189191

Fig 4. Inhibition of PRRSV JXwn06 replication by Ubc9.

Fig 4

(A) PRRSV titers in Ubc9-overexpressed MARC-145 cells. MARC-145 cells were transduced with the lentiviruses that were expressing GFP and Ubc9, respectively. The cells were infected with PRRSV JXwn06 at MOI of 0.01 at 24 h post-transduction, and the virus titers were then assayed by a microtitration infectivity assay at the indicated time points post-infection. Data are shown as means ± SD of three independent experiments (**p<0.01). (B) PRRSV RNA replication in Ubc9-overexpressing MARC-145 cells. MARC-145 cells were transduced with the lentiviruses that were expressing GFP and Ubc9, respectively. The cells were infected with PRRSV JXwn06 at MOI of 0.01 at 24 h post-transduction and collected at the indicated time points post-infection. The total cellular RNA was extracted and the mRNA levels of PRRSV N gene were determined by quantitative RT-PCR. Data are shown as means ± SD of three independent experiments (***p<0.001; ns, no significant). (C) SiRNA-mediated knockdown of endogenous Ubc9. MARC-145 cells were transfected with siRNA (siUbc9-1, siUbc9-2, siUbc9-3) and control siRNA (siCon). The cells were harvested at 48 h post-transfection and the cell lysates were probed with an anti-ubc9 antibody. The optical density ratios of Ubc9/β-actin in Ubc9 gene-silenced MARC-145 cells are shown with graphs. Data are shown as means ± SD of three independent experiments (***p<0.001). (D) The optical density ratios of Ubc9/β-actin in Ubc9 gene-silenced MARC-145 cells are shown with graphs. Data are shown as means ± SD of three independent experiments (***p<0.001). (E) PRRSV titers in Ubc9 gene-silenced MARC-145 cells. MARC-145 cells transfected with the siRNA (siUbc9-1) or control siRNA (siCon) for 48 h were infected with PRRSV JXwn06 at MOI of 0.01, and the virus titers were examined at the indicated time points post-infection. Data are shown as means ± SD of three independent experiments (*p<0.05; ***p<0.001; ns, no significant). (F) PRRSV RNA replication in Ubc9-silenced MARC-145 cells. MARC-145 cells transfected with the siRNA (siUbc9-1) or control siRNA (siCon) for 48 h were infected with PRRSV JXwn06 at MOI of 0.01 and collected at the indicated time points post-infection. The total cellular RNA was extracted and the mRNA levels of PRRSV N gene were determined by quantitative RT-PCR. Data are shown as means ± SD of three independent experiments (***p<0.001; ns, no significant). (G) PRRSV growth in the MARC-145 cells treated with GA. Data are shown as means ± SD of three independent experiments (***p<0.001; **p<0.01; ns, no significant).