Figure 4.

Knockdown of laboratory of genetics and physiology 2 (LGP2) potentiates grass carp reovirus (GCRV)-mediated activation of innate immune responses in Ctenopharyngodon idella kidney (CIK) cells. (A) Screening LGP2 interference sequences. Three siRNA sequences (s1, s2, and s3) along with the negative control si.C were transiently transfected into CIK cells. The cells were harvested for qRT-PCR at 24 h post-transfection to detect the transcription level of LGP2. (B) Examining the interference efficiency of the three siRNA in protein level. LGP2-Flag stable transfected CIK cells were transiently transfected with s1, s2, s3, and si.C in 6-well plates. Twenty-four hours later, cell lysates were prepared for IB using anti-Flag Ab. (C,D) Knockdown of LGP2 upregulates the protein levels of IRF3 and IRF7 induced by GCRV infection. CIK cells were transfected with s3 in 6-well plates. Twelve hours later, cells were infected with GCRV for 12 h and WB was conducted with anti-IRF3 and anti-IRF7 antiserums, respectively. (E,F) CIK cells were transfected with s3 and si.C, respectively, and treated or untreated with GCRV for 12 h. The cells were prepared for qRT-PCR to test the transcription levels of IFN1, IFN4, IFNγ2, and NF-κB2, respectively. Error bars indicate SD (n = 4). Asterisks indicate significant differences from control (*0.01 < P < 0.05; **0.001 < P < 0.01).