Skip to main content
. 2019 Aug 6;10:1788. doi: 10.3389/fimmu.2019.01788

Figure 3.

Figure 3

CREB mediates the early synergistic effect of cAMP. (A–C) Dominant negative CREB (A-CREB) inhibits mouse IL-10 promoter activity via the −376/−295 bp region. RAW264.7 cells were transfected with the indicated (B) or with the full (1,538 bp) (C) IL-10 promoter reporter plasmids and with A-CREB, or its control vector. The cells were treated with LPS (10 ng/ml) and isoproterenol (Iso, 1 μM) for 3 h (B), 8 h or 24 h (C). Luciferase reporter data represent three independent experiments and are expressed as mean ± SD of values normalized against renilla luciferase activity; *p = 0.015, ***p = 0.0002, ****p < 0.0001 for cells transfected with A-CREB compared to cells transfected with empty vector (two-way ANOVA followed by Sidak's post-test). The activity in resting cells was at least 3-fold lower than in treated cells. (D,E) CREB silencing blocks isoproterenol-stimulated IL-10 expression in LPS-treated macrophages. Stably CREB-silenced and shRNA-control RAW264.7 cells were incubated with LPS (10 ng/ml) ± isoproterenol (Iso, 1 μM) for 3 h. (D) CREB levels were analyzed by western blot. (E) IL-10 secretion to the medium was measured by ELISA and data representing eight independent experiments are expressed as mean ± SD; *p = 0.025, **p = 0.002, ****p < 0.0001 for silenced cells compared to control cells (two-way ANOVA followed by Sidak's post-test). All experiments were carried out twice with similar results.