iDCs pre-incubated with or without SP600125 and SB203580 (20 μM) for 1 h and infected with EV71 at a MOI of 5 for 24 h and the repilcation of EV71 was measured by TCID50. The results showed that the two inhibitors markedly inhibited EV71 replication (Figure  1A). Meanwhile, expression of EV71 VP1 protein in iDCs treated

with SP600125 selleck kinase inhibitor and SB203580 (20 μM) significantly reduced expression of EV71 VP1 protein at 4 h, 8 h and 24 h p.i., respectively (Figure  1B and C). Figure 1 The GSK1838705A supplier inhibitory effect of SP600125 and SB203580 on EV71 replication. (A) iDCs (3 × 105/well) pretreated with or without SP600125 and SB203580 (20 μM) for 1 h and infected with EV71 (MOI = 5) for 24 h, and culture supernatants were collected after infection to determine viral titers. (B and C) Western blot results of the supernatants and cell lysates of iDCs pre-incubated without or with SP600125 and SB203580 (20 μM) for 1 h and infected with EV71 (MOI = 5), using a specific antibody against VP1. The intensity of VP1 protein band quantitated by densitometric analysis and normalized to GAPDH. The data were expressed as mean ± SE from three independent experiments and analyzed by two-way ANOVA (***p

< 0.001). Activation of JNK1/2 and p38 MAPK during EV71 infection It has been reported that JNK1/2 and MI-503 mw p38 MAPK are phosphorylated during various virus infection [26, 27]. In order to assess whether activation of these G protein-coupled receptor kinase two MAPK signaling pathways occurred in EV71-infected iDCs, the degrees of total and phosphorylated JNK1/2 and p38 MAPK at 0 h, 1/2 h, 1 h, 2 h, 4 h, 8 h and 24 h p.i. were examined by Western blot. The results showed that EV71 infection enhanced not only mRNA levels of JNK1/2 and p38 MAPK (Table  1) but also their phosphorylation with prolonged infection. The phosphorylation of JNK1/2 reached its peak at 1 h p.i. (Figure  2A), while that of p38 MAPK reached its peak at 2 h and 24 h p.i., respectively(Figure  2C). Furthermore, the phosphorylation of JNK1/2 and p38 MAPK in EV71-infeced iDCs were significantly suppressed by pretreatment

with JNK1/2 and p38 MAPK inhibitor (SP600125 or SB203580) (Figure  2B and D). Therefore, JNK1/2 and p38 MAPK play important roles in EV71 replication cycle and phosphorylation of downstream molecules. Figure 2 EV71 infection stimulates activation of JNK1/2 and p38 MAPK. (A and C) Western blot analysis of cell lysates of iDCs infected with EV71 at a MOI of 5 at 0 h, 1/2 h, 1 h, 2 h, 4 h, 8 h and 24 h p.i. using antibodies against total or phosphorylated JNK1/2, p38 MAPK, as well as internal control GAPDH. (B and D) Western blot analysis of cell lysates of iDCs preincubated with SP600125 and SB203580 (20 μM) for 1 h and infected with EV71 at a MOI of 5 at indicated times using antibodies against total and phosphorylated JNK1/2, p38 MAPK, as well as internal control GAPDH.