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. 2018 Aug 9;293(41):15790–15800. doi: 10.1074/jbc.RA118.002721

Figure 2.

Figure 2.

Distal cis-elements regulate Il17a transcription in a STAT3-dependent manner. A fragment of the Il17a promoter was linked to a firefly luciferase reporter in the presence or absence of an additional sequence corresponding to a CNS element in the extended Il17a-Il17f loci. Plasmids were transfected into 5-day polarized primary murine Th17 cells along with a Renilla luciferase vector to control for transfection efficiency. Transfected cells were rested 14–18 h and then either left unstimulated or restimulated with anti-CD3 for 4 h (A) or 10 μg/ml cytokine (B and C) for 1 h. Luciferase readings were taken, and data are expressed as relative light units, normalized to Renilla luciferase activity. White boxes indicate predicted STAT-binding sites, and black boxes indicate predicted NF-κB-binding elements. *, p < 0.05, and #, p < 0.01 versus promoter alone under same manner of stimulation. C, luciferase activity of Th17 cells transfected with reporter constructs with (red X in box) or without (open box) mutated STAT-binding sites. Data (mean ± S.E. of duplicate samples) are representative of at least three independent experiments. *, p < 0.05, and #, p < 0.01 versus intact CNS construct. D, CD4+ T cells were isolated from Il17fThy1.1/Thy1.1 reporter mice and either assayed directly or grown under Th17 conditions for 6 days. Following Thy1 isolation and overnight rest, ChIP was performed with antibody directed against STAT3 or IgG in cells that were either left unstimulated, stimulated with IL-23 (3 ng/ml), or IL-23 plus IL-1β (10 μg/ml) for 1 h or PMA + ionomycin (PMA/I) for 4 h. Real-time PCR was performed on immunoprecipitated DNA using primer sets designed to detect the indicated CNS elements and promoter regions. The Socs3 and 16S ribosomal promoters were used as positive and negative controls, respectively. qPCR values were normalized to input DNA, and values representing relative STAT3 binding are expressed as n-fold increase versus naïve CD4. Results are the mean ± S.E. of two to five experiments. *, p < 0.05, and #, p < 0.01 versus STAT3 recruitment to the 16S ribosomal promoter (16Srp).