Fig. 3.

SOCS3 inhibits type I interferon-induced ISG expression to positively regulate the replication level of EV-D68. A–C HEK293T cells were transfected with the pCAGGS empty vector and the pCAGGS-SOCS3-flag plasmid for 24 ​h and then infected with EV-D68 (MOI ​= ​0.1) for 24 ​h. The cells were collected for qRT-PCR, Western blot, and detection of the TCID50 virus titer. D–F HEK293T cells were transfected into 10 ​μmol/L siRNA (siSOCS3) for 48 ​h; cells were then infected with EV-D68 (MOI ​= ​0.1) and incubated for 12 ​h. Cells were collected and subjected to qRT-PCR (D, E) and Western blot (F) to detect the RNA levels of SOCS3 and EV-D68 and protein levels of EV-D68. G–P SOCS3 was transfected into HEK293T and RD cells, and 24 ​h later, the cells were treated with IFN-β for 6 ​h. Subsequently, the cells were infected with EV-D68 (MOI ​= ​0.1) and incubated for 12 ​h. Cells were collected and subjected to qRT-PCR (G, H), Western blot (I, K), or viral titers (J, L) to detect EV-D68 RNA, protein, and titer levels. In addition, cells were collected and simultaneously subjected to qRT-PCR assays for ISG gene (RNaseL and ISG15) expression (M–P). Grayscale analysis of the Western blot results was performed using ImageJ software; three independent grayscale analyses were performed, and the results were averaged to ensure their reliability. qRT-PCR results are presented as the mean and standard deviation from three independent experiments. Statistical significance was analyzed using the Student's t-test (∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001, ​∗∗∗∗P ​< ​0.0001, ns, not significant).