FIG 3.

CDK1 positively regulates the replication of JEV. (A) HeLa cells viability assay at indicated concentrations of RO3306 or DMSO. (B) HeLa cells were treated with 5 μg/ml RO3306 or DMSO for 12, 24, and 36 h. Total cell lysates were collected for analysis by Western blotting with indicated antibodies. (C to E) HeLa cells were treated with 5 μg/ml RO3306 or DMSO for 2 h, and then cells were mock infected or infected with the WT virus at an MOI of 1. At 12, 24, and 36 hpi, viral titers (C), total viral mRNA expression (D), and JEV NS5 protein expression (E) were measured by plaque assay, Western blotting, and qRT-PCR, respectively. (F and G) HeLa cells were treated with 5 μg/ml RO3306 or DMSO for 2 h, and then cells were mock infected or infected with the WT virus at an MOI of 10 to examine the virus-cell binding (F) and virus entry into the cells (G) as mentioned in Materials and Methods. Protein levels of p-CDK1 were quantified by immunoblot scanning using image J software and normalized to the amount of GAPDH. All data were pooled from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, nonsignificant.