Fig. 4.

Pim1 signaling promotes EV-A71 replication through Hsp90β phosphorylation(unpublished data). a RD cells were treated with scramble/Hsp90β siRNA for 24h, the effects of Hsp90β knockdown were determined by RT-qPCR assay. The results are shown as the means, and ±error indicates the standard deviation (SD). Data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed Student’s t-test. b, c RD cells were treated with scramble /Hsp90β siRNA for 4h, then infected with EV-A71 at MOI of 1 for indicated time. The intracellular (b) and extracellular (c) viral RNA levels were detected by RT-qPCR assay. The results are shown as the means, and ±error indicates the standard deviation (SD). Data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed Student’s t-test. d, e knockdown of Hsp90β by siRNA or knockout by CRIPSR/Cas9 mediated gene editing in RD cells, and then cells were infected with EV-A71 at MOI of 1 for indicated time. The protein level of EV-A71 was determined by western blot. f RD cells were treated with Hsp90 inhibitors at different concentrations and infected with EV-A71 at MOI of 0.01 for 48h. The protein level of EV-A71 was determined by western blot. g Pim1-protein interaction network was predicted using online GENEMANIA program. h Pim 1 was overexpressed in 293T cells and cell lysate was collected for immunoprecipitation assay. The phosphorylation status of Hsp90 was detected by western blot. i Pim1 was overexpressed/knocked down in 293T cells. And cell lysate was collected for native page analysis. j WT RD cells and Hsp90β knockout (Hsp90β-KO) cells were pretreated with 2 μM PIM1 inhibitor SGI-1776 for 2 h, then the cells were infected cells with EV-A71 at MOI of 0.01 for 48h. Intracellular viral RNA level was determined by RT-qPCR. The results are shown as the means, and ±error indicates the standard deviation (SD). Data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed Student’s t-test. k Pim1 was overexpressed in WT/Hsp90β -knockout RD cells, and infected with EV-A71 at MOI of 1 for 12 h. The protein level of EV-A71 was determined by western blot