FIG 1.
ADAP2 is essential for the interferon response. (A) Knockdown of ADAP2 by use of two independent siRNAs reduced poly(I·C)-stimulated RIG-I-mediated IFN-β luciferase reporter activity in HEK293T cells. Gene knockdown efficiencies of the siRNAs as determined by Western blotting are shown in the inset. (B) Rescue of loss of IFN-β luciferase reporter activity in HEK293T cells caused by 3′-UTR-targeting siRNA-mediated endogenous ADAP2 silencing. The phenotype was rescued through overexpression of the ADAP2 ORF. (C) Knockdown of ADAP2 by use of two independent siRNAs reduced MDA5 overexpression-mediated IFN-β luciferase reporter activity in HEK293T cells. (D and E) ADAP2 knockdown reduced poly(I·C)-stimulated RIG-I-mediated IFN-α4 (D) and NF-κB (E) luciferase reporter activation. (F) ADAP2 knockdown attenuated ISG15 expression in Sendai virus-infected HEK293T cells as determined by qRT-PCR. (G) Knockdown of ADAP2 in human primary monocytes ablated SeV infection-induced IFNB1 transcript formation as determined by qRT-PCR analysis of viral RNA. (H) Knockdown of ADAP2 in HEK293T cells increased the VSV infection load as determined by plaque assay at 18 h postinfection. The luciferase values shown are means and SD for a representative experiment performed in triplicate. Reporter (firefly) luciferase values were normalized with a renilla luciferase internal control and expressed as fold induction values. RNA transcript loads were determined using qRT-PCR and expressed as fold changes. The qRT-PCR data were calculated by determining the relative threshold cycle (CT) values, based on the formula 2−(CT of target gene − CT of β-actin). The values for uninfected cells (F and G) were taken as 1. The statistical significance of differences in mean values was analyzed using unpaired two-tailed Student's t test, and P values of <0.05 were considered statistically significant (**, P < 0.01). si, siRNA; si-NT, nontargeting negative-control siRNA; EV, empty vector; UTR, untranslated region; ORF, open reading frame; VSV, vesicular stomatitis virus; SeV, Sendai virus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic internal control marker).