Figure 1.

DMXAA is a partial agonist for human STING interfering with agonist-mediated STING activation. (A) Fresh human PBMCs from three healthy donors (n=3) were stimulated with the indicated CDNs (10 µg/ml) for 24 h after 90 minutes of DMXAA (100 µg/ml) pretreatment. IFNβ, IFNγ, and IL-6 production was measured using ELISA. Data from each individual from two independent experiments are shown as bar graphs. (B, C) PMA-differentiated THP1 dual reporter cells were pretreated with DMXAA (100 µg/ml) for 90 min and stimulated with the indicated CDNs (10 µg/ml) for 12 or 24 h in three independent experiments. After RNA isolation and cDNA synthesis, the mRNA expression levels of IFNβ (B) and IL-6 (C) were measured using RT-PCR. 18S ribosomal RNA was used as an internal control. (B) IRF activity was determined by measuring secreted luciferase activity in the supernatants. (C) NF-κB activity was determined by measuring secreted embryonic alkaline phosphatase (SEAP) activity in the supernatant. Representative data from three independent experiments are shown as the mean ± SD of triplicates (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t test). (D) PMA-differentiated THP1 dual reporter cells were pretreated with DMXAA (100 µg/ml) for 90 min and stimulated with the indicated CDNs (10 µg/ml) for 3 h in three independent experiments. P-TBK1, TBK1, P-IRF3, IRF3, NF-κB and P-NF-κB levels in the cell lysates were detected using western blotting and the blots from a representative experiment were shown. B-actin was used as loading control. Fold induction levels for P-TBK1, P-IRF3 and NF-κB relative to total TBK1, IRF3 and NF-κB from three independent experiments were plotted as mean ± SD (n=3, biological replicates) (*p < 0.05, **p < 0.01, Student’s t test).