Fig. 5.

SOCS3 inhibitor inhibited EV-D68's RNA and protein expression. A Schematic representation of SOCS3 truncating mutants. B–E Wildtype SOCS3, SOCS3 ΔKIR, SOCS3 ΔSH2-Q, SOCS3 ΔSH2-H, and SOCS3 ΔBOX were expressed in HEK293T cells and RD cells for 24 ​h, then infection with EV-D68 for 24 ​h, and the cells were collected. The RNA and protein levels of EV-D68 were detected by qRT-PCR (B,C) and Western blot (D,E). D Peptides, inhibitor-1 and inhibitor-2, were designed to specifically bind to the KIR domain of SOCS3 to inhibit its function. G Transfection of inhibitor-NC, inhibitor-1, and inhibitor-2 in HEK293T cells for 24 ​h. Cells were collected for the CCK8 assay to detect cell viability. H HeLa cells were transfected with the pCAGGS-SOCS3-flag plasmid, inhibitor-2 plasmid, or co-transfected with the above two plasmids. After 24 ​h, the cells were fixed, and the intracellular localization of SOCS3 and inhibitor-2 was detected by immunofluorescence. The line-scanning plots were used to quantify florescence intensity. I-L Transfection of inhibitor-NC, inhibitor-1, and inhibitor-2 in HEK293T cells and RD cells for 24 ​h, then 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 (I, J) and Western blot (K, L) to detect EV-D68 RNA protein levels. 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).