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. 2019 Aug 6;10:1788. doi: 10.3389/fimmu.2019.01788

Figure 4.

Figure 4

Cooperativity between tandem CRE sites is essential for cAMP-stimulated IL-10 promoter activity. RAW264.7 macrophages were transfected with a luciferase reporter regulated by four repeats of either CRE1 or CRE2 or a consensus CRE (A), two repeats of the −362/−324 bp region with either the WT sequence, CRE1 mutant, or CRE2 mutant (B), or the full (−1,538 bp) IL-10 promoter, either wt, mutant in CRE1 (mCRE1), mutant in CRE2 (mCRE2) or a double mutant (mCRE1+2) (C). Site mutation is shown by red color. The cells were incubated with isoproterenol (Iso, 1 μM) and/or LPS (10 ng/ml) for 3 h (A,B) or with both stimuli for the indicated time (C). Luciferase reporter data represent three independent experiments and are expressed as mean ± SD of values normalized against renilla luciferase activity, relative to unstimulated (A,B) or LPS-stimulated (C) control cells; (A,B) **p = 0.001, ****p < 0.0001 for cells treated with isoproterenol (± LPS) compared to control cells (two-way ANOVA followed by Dunnett's post-test). (C) Error bars represent the sum of SD (%) of both values in the ratio. ****p < 0.0001 for mutants compared to wt (two-way ANOVA followed by Dunnett's post-test). The experiments were carried out 3 times with similar results.