Figure 5

Specific knock down of DUSP6 increases the phosphorylation of ERK1/2, reduces apoptosis, and promotes IBV replication. Vero (A), H1299 (B), DF-1 (C) cells were transfected with non-target siRNA (sic), siDUSP6-1 or siDUSP6-2 for 36 h, followed with IBV infection. Cells were harvested at 20 and 24 hpi and subjected to cell lysis or RNA extraction. The knock down effect of DUSP6 was analyzed by quantitative real time RT-PCR (low panel). The level of p-ERK1/2, ERK1/2, PARP, Bcl-2, Mcl-1, IBV N, and β-actin were checked with Western blot analysis. The experiments were repeated in triplicate and the representative data are shown. The signal of protein bands was determined by Image J software. The intensities of p-ERK1/2 or p-ERK2 were normalized to total ERK1/2 or total ERK2, the intensities of Bcl-2, Mcl-1, IBV N were normalized to β-actin, and the intensities of PARP-C were normalized to PARP-FL. The ratio of p-ERK1/2, p-ERK2, Bcl-2, Mcl-1, PARP-C, IBV N in siDUSP6 transfected cells to sic transfected cells were shown as (siDUSP6:sic). The bar graphs in the low panels show means ± SD of three independent determinations of relative expression of DUSP6 mRNA. P values were calculated by Student test. ****P < 0.0001 (highly significant).