Figure 6. Tnf-α/NF-κB and Notch crosstalk leads to Hes1-mediated Pparg inhibition.

(A) Pparg mRNA expression in 2- and 5-month-old Kras^G12DTnfa^ΔPdx and Kras^G12DIkk2^ΔPdx pancreases. Data were normalized to Kras^G12D pancreases. Data are shown as mean + SD; n = 6. ***P < 0.001. The experiment was performed in duplicate. (B) Tnf-α stimulation (1 ng/ml) induced downregulation of Pparg in Kras^G12DTnfa^ΔPdx PanIN cell lines. (C) ChIP was performed on Kras^G12D cells using anti-Hes1 or a control IgG. Precipitated DNA was amplified by real-time PCR using primers specific for Pparg. (D) siRNA knockdown of Hes1 upregulated Pparg and Cebpa expression in Kras^G12D PanIN cells. (E) Kras^G12D and Kras^G12DTnfa^ΔPdx PanIN cells were cotransfected in duplicate with a Pparg reporter construct containing 1,500 bases of the proximal Pparg promoter (full length) and a Hes1 expression plasmid or empty vector control. Twenty-four hours after transfection, cells were analyzed for luciferase activity. (F) Transfection of Kras^G12DTnfa^ΔPdx PanIN cells as described in E with a full-length Pparg reporter construct or a construct with a truncated Hes1-binding sequence. All data are shown as mean + SD from duplicate transfections and are representative of 3 independent experiments.