14-3-3ε knockdown and difopein treatment decrease HP-PRRSV infection in PAMs. A Results of qPCR for detection of 14-3-3ε after knockdown. PAMs were transfected with siRNA. The cells were collected at 24 h post-transfection, and total RNA was prepared for detecting the mRNA levels of 14-3-3ε. B Results of qPCR for analysis of TA-12 replication after 14-3-3ε knockdown. PAMs were transfected with siRNA and inoculated with TA-12 at 24 h post-transfection. The cells were collected, and total RNA was extracted. Viral loads were evaluated by absolute qPCR targeting the N gene of TA-12. C The cytotoxicity of difopein in PAMs was determined by the CCK-8 assay. PAMs were inoculated with TA-12 or CH-1R and incubated for 1 h, after which the medium was replaced with maintenance medium containing 0.02 or 0.08 μg/mL difopein. The cells were collected 24 h later, and total RNA was extracted. Viral loads were evaluated by absolute qPCR targeting the N gene, and viral proteins were detected by Western blot analysis of TA-12- (D) CH-1R- (E) infected cells. F Therapeutic role of difopein in TA-12 infection. PAMs were infected with TA-12 and treated with 0.02 or 0.08 μg/mL difopein at 24 hpi. The cells were incubated for another 12 h and then harvested for RNA extraction and qPCR analysis.