Figure 3.

SOCS1 counteracted the anti-PEDV activity of IFN-λ. (A) The knockdown efficiency of shSOCS1 was determined by Western blotting (left panel); β-actin was used as a loading control. The SOCS1 bands were quantified using ImageJ software as normalized to β-actin (right panel). (B) Knockdown of SOCS1 decreased PEDV replication. Vero E6 cells were treated with 100 ng/mL of IFN-λ and transfected with NC or shSOCS1s, followed by infection with PEDV (MOI = 0.1). After 36 h, cell culture supernatants were harvested for virus titration. (C) Silencing of SOCS1 increased IFN-λ signaling induced by IFN-λ stimulation. Vero E6 cells were stimulated with IFN-λ 12 h after transfection with shSOCS1 #1, shSOCS1 #2, shSOCS1 #3, or scrambled control shRNA, and then the cells were collected for RT-qPCR analysis of ISG15, IFIT1, or MxA expression relative to that of GAPDH after 24 h of stimulation. (D) Transient expression of SOCS1 in Vero E6 cells. SOCS1 was cloned and expressed in the eukaryotic expression vector pCAGGS-HA. The overexpression efficiency of SOCS1 in Vero E6 cells was confirmed by IFA. (E) Transient expression of SOCS1 enhanced PEDV infection in Vero E6 cells. Vero E6 cells were stimulated with IFN-λ 12 h after the transient expression of SOCS1 promoted PEDV infection. Vero E6 cells were transfected with SOCS1-HA for 24 h and then infected with PEDV (MOI = 0.1). PEDV infection was determined by measuring PEDV titers. (F) Transient expression of SOCS1 disrupted the IFN-λ antiviral response. Vero E6 cells were stimulated with IFN-λ after being transfected with SOCS1 for 24 h. The cells were collected for RT-qPCR analysis of ISG15, MxA, and IFIT1 expression relative to that of GAPDH after 24 h of stimulation. Error bars, mean ±SEM. (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, and NS, not significant.