Fig 2.

siRNA knockdown of SHFL enhances PRRSV infection in MARC-145 cells and PAMs. (A–C) MARC-145 cells were transfected with Monkey-siSHFL or siNC for 48 h. Cells were infected with rTA-EGFP (MOI = 0.01, 0.1, 1) and harvested at the indicated time points for Western blot analysis, and culture supernatants were harvested for virus titration. The replication kinetics of rTA-EGFP were monitored by EGFP signal under fluorescence microscopy (scale bar, 100 μm) (A). The viral titers in the supernatant were determined by the TCID₅₀ assay in MARC-145 cells (B). The expression of viral proteins (nsp4, nsp9, and N) and endogenous SHFL was evaluated by Western blot analysis (C). (D–F) PAMs were transfected with siNC or siSHFL (Pig-siSHFL-1–3) for 48 h and then infected with NL1207 (MOI = 0.3). PRRSV replication was evaluated by IFA using a mAb against N protein (scale bar, 100 μm) (D), Western blot analysis of viral nsp9 (E), and virus yields in culture supernatants (F). Data presented in panels B and F are means ± SD of three replicates. Statistical significance was analyzed using multiple unpaired t-tests. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant.