Figure 3: ICER is induced by IL-6 via STAT3 and, binds to the IL-17A promoter.

(a–c) CREM and β-actin expression in (a) B6 naive CD4⁺ T cells cultured in the presence of indicated stimulations on day2, (b) Th17-polarized B6 naive CD4⁺ T cells cultured in the presence of STAT3 inhibitor (STA21; 0, 10 and 30 μM) or SMAD3 inhibitor (SIS3; 0, 1 and 3 μM) on day3, and (c) Th17-polarized naive CD4⁺ T cells from indicated strains in the presence or absence of rIL-6 on day3 was measured by western blotting. Data are representative of 3–4 experiments. (d,g) Schematic representations of (d) CRE (−116/−53, +573/+580 and +639/+646) and of Stat3-binding site (+586/+594) on the p2 ICER promoter, and (g) CRE (−116/−109) on the IL-17a promoter are shown. (e) The full-length ICER promoter region (Full) or a version containing the STAT3-binding site-directed mutation (Δ+586) were transfected to Th17-polarized or Th0-polarized cells. The ratio of reporter activities in those Th17-polarized cells on those Th0-polarized cells was shown (*P<0.05; mean±s.e.m.; n=3). (f,i) Binding of (f) FLAG/ICERγ to the CRE or (i) RORγt to the ROR-binding element in Th17-polarized CD4+ T cells from ICER/CREM^−/− mice after indicated vector transfection. A comparison to the binding of control Rabbit IgG Ab and sample input. Data are representative of three experiments. For f, the first intron of ICER was used as a negative control for ChIP enrichment. (h) IL-17a promoter activity of Th17-polarized ICER/CREM^−/− naive CD4⁺ T cells was shown. Either full-length mouse IL-17a promoter or a version containing the CRE site-directed mutation (Δ-116) were transfected (**P<0.01; mean±s.e.m.; n=3). See Supplementary Fig. 3 for uncropped scans of the western blot.