Fig. 3.

Mda5 expression is stable and the expression is dependent on STAT1. a BMDMs were treated with or without IFN-α for 6 h, then DRB (20 g/mL) and actinomycin D (5 g/mL) were added. Mda5 expression was then measured by RT-PCR at the indicated time points. Cell viability was 95% for all culture conditions. As a control, we also determined the half-life of c-myc (n = 3). b Similar experiment to that in (a), but LPS was used instead as the Mda5 activator (n = 3). c BMDMs from Stat1 knockout mice and the corresponding wild-type counterparts were isolated and stimulated with IFN-γ, IFN-α, or LPS for 6 h. Mda5 expression was analyzed by RT-PCR (n = 4). d Similar experiment to that in (c), but using electroporated poly(I:C) as the activator (n = 4). e Similar experiment to that in (c), but using IFN-γ as the activator and analyzing Ifn-β expression (n = 4). f BMDMs were incubated in the presence or not of 5 μg/mL of CHX for 1 h, then IFN-α, LPS, or poly(I:C) was added and incubated for 3 h and Mda5 was determined (n = 4). g Similar experiment to that in (f), but Tnf-α was determined (n = 4). Each experiment was performed in triplicate, and the results are shown as the mean ± SD. **p < 0.01, ***p < 0.001, and p < 0.0001 in relation to each stimulated sample compared to controls in each experiment. Data were analyzed using the unpaired Student's t test.