FIG 4.
Inhibition of seasonal IAV replication in human moDCs by pretransfection with a cIAV DsiRNA swarm. Human moDCs from four different blood donors were separately mock transfected (control, UV IAV, or no-siRNA bars) or pretransfected with the indicated siRNAs (10 nM) for 21 h. Cells were subsequently infected with the indicated live or with UV-irradiated seasonal IAVs at an MOI of 1. (A) At 24 h p.i. cells from different blood donors were pooled, and the IAV M1 RNA expression was determined by qRT-PCR from isolated total cellular RNA samples. The values were normalized against β-actin gene-specific mRNA, and relative IAV M1 RNA levels were calculated by the ΔΔCT method using untreated 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 from samples of nontransfected cells (no siRNA; boxed bars). *, P < 0.05; **, P < 0.01. (B) Western blot analysis for the expression of viral PB1, NP, M1, and NS1 proteins and β-actin in siRNA/DsiRNA transfected moDCs infected with the indicated seasonal IAVs. Cells were collected at 24 h p.i., 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.