Fig 7. Silencing of MINK and inhibition of p38 MAPK phosphorylation reduces translation efficiency of EV71 IRES.

(A) Schematic diagram of the bicistronic construct containing the Renilla luciferase (RLuc) and firefly luciferase (FLuc) genes controlled by the cytomegalovirus (CMV) promoter and 5’ UTR of EV71–26M [67], respectively. (B) Effect of knockdown of MINK on EV71 IRES activity. RD cells were pre-treated with MINK or scrambled siRNA. Three days after transfection, the bicistronic construct was then transfected into the cells. Luciferase activity was measured 24h after transfection. Amantadine, an inhibitor of EV71 IRES [33], was added to untreated cells to serve as negative control for IRES activity. Untreated cells that were transfected with the bicistronic construct were used as positive control. The FLuc/RLuc ratio for each sample were normalised to the FLuc/RLuc ratio of untreated control. Dose-dependent reduction in relative translation efficiency of the IRES was observed in MINK siRNA-treated cells. Error bars represent standard deviation of triplicate data sets. Statistical analyses were performed using one-way ANOVA with Dunnett’s test (Graphpad software). *P < 0.05, **P < 0.01 and *** P < 0.0001 versus untreated control. (C) Effect of p38 MAPK inhibition on EV71 IRES activity. Relative translation efficiency was determined as the ratio of FLuc to RLuc for each sample and the FLuc/ RLuc ratio for each sample were normalised to the FLuc/RLuc ratio of DMSO control, expressed as percentage. Error bars reflect the standard deviation of triplicate data sets. Transfected cells with the bicistronic construct without drug treatment (DMSO control) was used as positive control. Error bars represent standard deviation of triplicate data sets. Statistical analyses were performed using one-way ANOVA with Dunnett’s test (Graphpad software). *P < 0.05, **P < 0.01 and *** P < 0.0001 versus 1.0% DMSO control.