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. Author manuscript; available in PMC: 2010 Feb 3.
Published in final edited form as: Cancer Cell. 2009 Feb 3;15(2):114–123. doi: 10.1016/j.ccr.2008.12.018

Figure 2. Stat3 and NF-κB/p65 synergistically enhance IL-23 expression by directly binding to the IL-23/p19 promoter.

Figure 2

(A) Stat3 upregulates expression of IL-23/p19 mRNA in CD11b+ myeloid cells freshly isolated from B16 tumors. Shown are the results from one of three independent experiments analyzed by real-time PCR, normalized to 18S rRNA. (B) Over expression of constitutively active Stat3 mutant (Stat3C) activates transcription by IL-23/p19 promoter. The fragment from the mouse promoter of IL-23/p19 (sequence from -1159 to +160) was cloned into pGL3 vector with luciferase reporter gene. Dual-luciferase activity was determined 24 h after transfection of various sets of expression vectors into 3T3 fibroblasts. (C) Stat3 and NF-κB/p65 bind directly to the p19 promoter. Top: Stat3 silencing downregulates the activity of IL-23/p19 promoter. Dual-luciferase activity was measured in lysates of B16 cells 24 h after transfection with various concentrations of Stat3 siRNA or with scrambled RNA control. Bottom: Both Stat3 and NF-κB/p65 transcription factors are required for the transcriptional activity of IL-23/p19 promoter. Stat3 siRNA, NF-κB/p65 siRNA or both were transfected into B16 cells together with IL-23/p19 promoter-luciferase construct. Dual-luciferase activity was measured as described above. Data shown are mean values ± SD from experiments performed in triplicates.