Figure 4

Pharmacological inhibition of DUSP6 promotes ERK1/2 signaling, reduces apoptosis, and facilitates IBV replication. Vero (A), H1299 (B), and DF-1 (C) cells were pre-incubated with BCI (10 μM) or DMSO for 1 h, followed by IBV infection. 10 μM BCI or DMSO was present throughout the experiment. Mock infection was included as another control group. Cells were harvested at 20 and 24 hpi and subjected to cell lysis or RNA extraction. The level of p-ERK1/2, ERK1/2, PARP, Bcl-2, Mcl-1, IBV N, and β-actin were checked by Western blot analysis (upper panels). The expression of DUSP6 was analyzed by quantitative real time RT-PCR (low panels). 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 PARP-C and IBV N in BCI treated cells to DMSO treated cells are shown as PARP-C (BCI:DMSO) and IBV N (BCI:DMSO). The bar graphs in the low panels show means ± SD of three independent determination of relative expression of DUSP6 mRNA. P values were calculated by Student test. ***P < 0.001, ****P < 0.0001 (highly significant).