Figure 5. IRF3 regulates GLI3 by directly binding to IRF3 binding sites.

(A) Schematic diagram of ~ 3000 bp upstream of ATG located in exon 2 of GLI3. Several candidate IRF3 binding sites (BS) were identified. (B) Untreated MM6 cells (10 × 10⁶) were lysed and a chromatin immunoprecipitation (ChIP) assay was performed followed by qPCR using three primer sets for the areas containing IRF3 binding sites upstream of ATG of GLI3. (C) MM6 cells (10 × 10⁶) were treated with 10 μg/ml Poly(I:C) for 1h prior to ChIP and qPCR. (D) Primary human monocytes (D3) were treated with 10μg/ml Poly(I:C) for 1h prior to ChIP and qPCR. (E) Primary human monocytes (D3) were challenged with 10 μg/ml Poly(I:C) for 12h prior to western blot analysis. (F) U937 cells (4 × 10⁶) were transfected with either IRF3 expression construct or empty vector along with pGL4.1_GLI3 promoter construct and pGL4.7 Renilla plasmid as described in the methods section. After 48 hr, cells were harvested and lysed and lysates were used to quantify luciferase activity. These experiments were performed at least 3 times with similar results.