FIG 9.
Inhibition of H3N2 IAV replication by pretransfection with cIAV DsiRNA swarm in mouse KO cells. Mouse wt cells, IRF3/IRF7 double-KO cells (IRF3/7 KO cells), NF-κB RelA/c-Rel/Nfkb1 triple-KO cells (NF-κB KO cells), and IFN-α/β receptor 1 KO cells (IFNAR1 KO cells) (in 12-well plates; 5 × 105 cells/well) were mock transfected (control or no-siRNA bars) or pretransfected with the indicated control and IAV-specific siRNA/DsiRNAs (10 nM). After 21 h of incubation, cells were infected with H3N2 IAV (A/Udorn/307/1972) at an MOI of 1 for an additional 24 h. Cells were then collected for RNA isolation and quantitative RT-PCR analysis and for Western blot analysis. (A) The values of RT-PCR analyses were normalized against β-actin gene-specific mRNA, and the relative IAV M1 mRNA level was calculated by the ΔΔCT method using untreated control cells as a calibrator. The means (±SD) of three parallel analyses are shown. Data are representative of three individual experiments. Statistical significance was determined against results of samples of nontransfected cells (boxed bars). *, P < 0.05. (B) Western blot analysis for the expression of IAV proteins PB1 and NP and cellular β-actin in siRNA/DsiRNA transfected cells after the infection of H3N2 IAV. Cells were collected at 24 h after infection, and whole-cell lysates were prepared. Cellular proteins (30 μg/lane) were separated by 10% SDS-PAGE, followed by Western blot analysis with the indicated antibodies. One representative experiment of three independent experiments is shown.