<p>Gene knockdowns using siRNA specific to target genes in hAECs. The experiments were carried out using target gene-specific siRNA and the control nonspecific siRNA, which were reverse-transfected into hAECs at the concentration of 100 nM. The data presented are the corresponding mRNA levels at 6 hr after electroporation (fold change, in logarithmic scale). (A), knockdown of RelA, IRF3, RIG-I and IKKγ. (B), knockdown of TNFAIP3/A20, NFKBIA/IκBα, ISG56 and IFNβ. The mRNA levels of the indicated genes (at top) were determined by RT-PCR. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093396#s3" target="_blank">Results</a> were represented as normalized fold change of expression compared to control non dsRNA-induced cells transfected with scrambled (non-target control) siRNA. Gray bars, experiment (dark, no dsRNA stimulation; light, 4 µg dsRNA); red lines, model simulation. 95% confidence intervals of experimental data (based on 3 replicates) are distorted by the logarithmic scale. In linear scale, the relative error rate is approximately 10%. Please notice that in IFNβ and ISG56 charts, the model values at time 0 are equal to 0, which is impossible to depict on the log scale. (C), Effect of RIG-I knockdown on NF-κB and IRF3 dependent gene expression. hAECs were transfected with siRNA to RIG-I or control siRNA (Con). Left panel, effect of RIG-I siRNA on RIG-I expression. Note that dsRNA induced RIG-I expression is largely inhibited in hAECs transfected with RIG-I siRNA. Middle panel, effect of RIG-I knockdown on NF-κB-dependent TNFAIP3/A20 gene expression. dsRNA induces TNFAIP3/A20 expression in RIG-I knockdown cells. Right panel, effect of RIG-I knockdown on IRF3-dependent gene expression. RIG-I knockdown significantly blunts IRF3-dependent ISG56 expression. We conclude from these data that RIG-I is primarily coupled to IRF3 signaling in hAECs. (D), Effect on RIG-I expression in murine MEF cells with both IRF3 and IRF7 genes knocked down. RIG-I is down-regulated, which suggests IRF7 may play a key role in RIG-I up-regulation. Compare with RIG-I results in Panel A where the IRF3 knock-down does not seem to down-regulate RIG-I expression.</p

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