FIGURE 2.

RIG-I signaling systematically increases with dsRNA length. A, RNA used for transfection was extensively purified and quality controlled. A graphical representation of capillary electrophoresis of purified positive (+) and negative (−) strand in vitro transcripts is shown. The band marked with the black arrowhead corresponds to the internal calibrator of the system. B, Huh7.5/RIG-I cells were stimulated with dsRNA of 100 bp (ds100, squares) or 400 bp (ds400, circles) length, and ISG56 promoter reporter activity was determined. Dose-response was standardized and fitted using Hill regression. Data points were combined from two independent repetitions of the experiment. C and D, MEFs were transfected with ds100 or ds400 and ISG56 promoter reporter activity was assessed. MEFs were derived from mice without (C) or with (D) a homozygous deletion of the rig-I or mda5 gene, respectively (as indicated). E, ISG56 promoter-reporter activity was assessed in Huh7.5/RIG-I cells upon stimulation with equal numbers of dsRNA molecules of increasing length. Independent response curves for three different concentrations are given (as indicated). Paradoxical behavior for unphysiological molecule numbers is shown as a dashed line. F, the three absolute response curves from E were standardized to the respective maximum signal (100%), and data points were averaged (±S.D.). A Hill model was fitted to the data, which was significantly more accurate than standard mass action kinetics (p < 0.0001 in extra sum-of-square F-test). dsRNA lengths required to elicit 10%, 50%, and 90% of the maximum signal were calculated from the regression and are given with the respective 95% confidence intervals in the inset.