Innate immunity responses to siRNA pools and single-site siRNA molecules.

<p>HaCaT and U373MG cells were transfected with either 1 (light grey) or 10 (dark grey) pmol of the indicated siRNA molecules; 1 pmol of 88-bp dsRNA (black bar) or water (control, grey bar); or left untreated (control, white bar). The expression levels of <i>IFN-β</i> (<i>A</i>), <i>IFN-λ1</i> (<i>B</i>) or <i>ISG54</i> (<i>C</i>) were assessed by qRT-PCR 8 h, 24 h or 48 h post transfection. Values were normalized to the <i>GAPDH</i> housekeeping gene and shown on a logarithmic scale. The mean values+S.D. of at least two independent experiments, each with a minimum of three biological replicates, are shown. Data were compared by Mann-Whitney U-test. The statistical significance is indicated as (×) p < 0.01 or (¤) p < 0.05 against the controls; (**) p < 0.01 or (*) p < 0.05 against a group of comparison; (#) p < 0.01 against all other transfections. UL29GD, GD-digested anti-<i>UL29</i> siRNA pool; UL29HD, HD-digested anti-<i>UL29</i> siRNA pool; UL29L, 27-nt chemically synthesized anti-<i>UL29</i> siRNA; UL29S, 21-nt chemically synthesized single anti-<i>UL29</i> siRNA. No significant signal was detected for IFN-λ1 mRNA in U373MG cell culture (B, right panel) if no dsRNA was applied (non- and mock-transfected cells).</p

Inhibition of HSV-1 replication by <i>UL29</i>-specific siRNA molecules.

<p>HaCaT and U373MG cells on 96-well plates were transfected with 10 pmol of indicated siRNA molecules or water. After 4 h the cells were infected with 1000 pfu of HSV-1 and incubated for 44 h. (<i>A</i>) Dilutions of the HaCaT and U373MG supernatant, collected 48 h after transfection (44 h post infection) were assayed for released virus by plaque formation on Vero cell culture. (<i>B</i>) The relative expression of the target HSV-1 gene <i>UL29</i> was measured by qRT-PCR from samples of the infected cultures. Values were normalized to the <i>GAPDH</i> housekeeping gene and shown on a logarithmic scale. The mean values+S.D. are shown for six replicates. Data were compared by Mann-Whitney U-test. The statistical significance is indicated as (×) p < 0.01 and (¤) p<0.05 against the controls; (*) p < 0.05 against a group of comparison; (#) p < 0.01 against all other siRNA transfections. NT, no transfection; mock, transfection with water; eGFPGD, anti-eGFP siRNA pool; eGFPS, single-site anti-eGFP siRNA; UL29GD, GD-digested anti-UL29 siRNA pool; UL29HD, HD-digested anti-UL29 siRNA pool; UL29L, 27-nt single-site anti-UL29 siRNA; UL29S, 21-nt single-site anti-UL29 siRNA.</p