Fig. 5

The effect of MAP4K4 knockdown on EV71 replication. a qRT-PCR and, b Western blotting were performed to assess the expression of MAP4K4 in response to EV71 infection in RD cells. RD cells were infected with or without EV71 at 5 MOI at the indicated time points. The right panel in (b) shows that the MAP4K4 protein level normalized to GAPDH is expressed as a fold change compared with the mock infection (24 h) group, as quantified using Image J software. c The effect of MAP4K4 knockdown on the virus titer was determined at 24 h p.i. by plaque assays. RD cells were transfected with the LV-shcontrol or LV-shMAP4K4-3 vector for 48 h, followed by infection with EV71 (MOI = 5) for 24 h. Total virus was collected from infected cells and culture supernatants and used in plaque assays. The virus titers in the cultures are expressed as PFU per milliliter. d Viral RNA and MAP4K4 mRNA expression and (e) VP1 protein and MAP4K4 protein expression following MAP4K4 knockdown were determined by qRT-PCR and Western blotting, respectively. The LV-shcontrol or LV-shMAP4K4-3 vector was transfected into RD cells for 48 h, followed by EV71 infection (MOI = 5) for 24 h. The right panel in (e) shows that the VP1 and MAP4K4 expression levels normalized to GAPDH are expressed as fold changes compared with the LV-shcontrol-transfected group, as quantified using Image J software. f The effect of MAP4K4 silencing on cell viability was assessed by MTT assay. RD cells were transfected with the LV-shcontrol (140 ng) or LV-shMAP4K4-3 (140 ng) vector for 48 h and then infected with or without EV71 (MOI = 5) for 24 h. Cell viability is expressed as percentages of the LV-shcontrol-transfected and mock-infected cells, which were used as controls. *p < 0.05; **p < 0.01; ***p < 0.001; ns no significance