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. 2019 Dec 18;12(1):2. doi: 10.3390/v12010002

Figure 7.

Figure 7

SOCS5 negatively regulates type I IFN signalling and facilitates FHV-1 replication. (A–D) Overexpression of SOCS5 blocked IFN- induced signalling cascades and promoted FHV-1 replication. F81 cells were transfected with p3×Flag-CMV-SOCS5 or empty vectors for 24 h, followed by FHV-1 infection (MOI = 1 for p-STAT1 expression and ISGs mRNA analysis, and MOI = 0.1 for virus titres analysis) for 24 h or IFN-β stimulation (200 ng/mL) for 30 min. Then, the expression levels of p-STAT1 and STAT1 were identified by WB analysis (A). GAPDH served as an internal control. Total RNA from another sample was extracted for qRT-PCR to detect the expression of ISG15, Viperin and IFITM1 (B). The relative expression levels of these genes were calculated by normalising to that of cellular 18S RNA. The supernatants and cells were collected for testing virus titres via the plaque assay (C) and virus copies by absolute quantification PCR (D), respectively. (E–I) Knockdown of endogenous SOCS5 enhanced IFN-elicited signalling cascades and suppressed FHV-1 replication. F81 cells were transfected with three siRNAs targeting SOCS5 for 48 h. The silencing efficiency was evaluated through testing cellular SOCS5 by western blot (E). siSOCS5#1 was selected for transfection into F81 cells. Thirty-six hours after transfection, the cells were infected with FHV-1 (MOI = 1 for p-STAT1 expression and ISGs mRNA analysis, and MOI = 0.1 for virus titres analysis) for 24 h or stimulated by IFN-β (200 ng/mL) for 30 min. Then, the protein levels of p-STAT1 (F) and the mRNA expression of ISG15, Viperin and IFITM1 (G) were detected according to the method described above. Virus titres (H) and virus copies (I) were determined via the plaque assay or absolute quantification PCR, respectively. Empty vectors and siNC groups served as negative controls. All samples were independently repeated three times, and data are representative of three independent experiments. The significant differences are indicated as follows: NS > 0.05, * p < 0.05, ** p < 0.01.