Figure 4.

Kinetics of the suppressive effect of HMMX in THP1 dual reporter cells. (A) PMA-differentiated THP1 dual reporter cells were stimulated with various concentrations of HHMX (1, 3, 10, or 30 µg/ml) together with increasing concentrations of c-di-AMP (3, 10, or 30 µg/ml) for 24 h in five independent experiments. IRF activity was determined by measuring secreted luciferase activity in the supernatants Data are shown as the mean ± SD of five independent experiments (n=5, biological replicates). IFNβ concentration in the supernatants was determined using ELISA. Data are shown as the mean ± SD from three independent experiments (n=3, biological replicates) (*p < 0.05, **p < 0.01, ****p < 0.0001, One-way ANOVA with Sidak’s multiple comparison test). (B) PMA-differentiated THP1 dual reporter cells were stimulated with HHMX (30 µg/ml) at indicated the time points (−3, −1.5, 0, 1.5, or 3 h) together with c-di-AMP (30 µg/ml) for 24 h in three independent experiments. IRF activity was determined by measuring secreted luciferase activity in the supernatants. Representative data from three independent experiments are shown as the mean ± SD of triplicates from one experiment (n=3, technical replicates). IFNβ concentration in the supernatants was determined using ELISA. Representative data from three independent experiments are shown as the mean ± SD of triplicates from one experiment (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t test.) (C) NF-κB activity was determined by measuring secreted embryonic alkaline phosphatase (SEAP) activity in the supernatant. Data are shown as the mean ± SD from three independent experiments (n=3, biological replicates) (*p < 0.05, **p < 0.01, Student’s t test.).