FIGURE 1:

(A) The structure of the COL1A2 far-upstream enhancer (FUE) showing the proximal promoter (PP), three of the five DNAse hypersensitive sites (HS3–5), the critical AP-1 element in HS4, and the two putative STAT3 sites (arrows indicate DNA strand). (B) Reporter gene analysis of enhancer activation in normal (n = 3) and SSc (n = 3) lung fibroblasts treated with the JAK/STAT inhibitor AG490. The structure of the two DNA constructs used is shown at the top. The WT construct contains all elements of the FUE and PP fused to the firefly luciferase gene, while in ΔHS4 the HS4 region has been deleted to inactivate the enhancer. The results are normalized using a Renilla luciferase transfection control. (C) DNA gel showing the results of a ChIP assay for phosphoSTAT3 near the HS4 region in SSc lung fibroblasts treated with IL6 plus the soluble receptor α(IL6xRα) and SD1029. (D) Western blot of nuclear extracts from the same cells used in C, showing nuclear phosphoSTAT3 levels. TATA binding protein (TBP) was used as the house keeping gene. (E) ChIP analysis using qPCR for the binding of STAT3, JunB, and RNA polymerase II, in the HS4 region of SSc fibroblasts after treatment with SD1029, STAT3 siRNA (siSTAT3), or nontargeting siRNA (siNT). * = p < 0.05.