Figure 4
From: The PRKD1 promoter is a target of the KRas-NF-κB pathway in pancreatic cancer
Mapping of the NF-κB site relevant for KRasG12V-mediated PRKD1 promoter activation.
(A) Cells were transfected with vector control or p65 as well as full-length or indicated truncated versions of the PRKD1 promoter luciferase reporter and renilla-luciferase reporter, as indicated. 24 hours after transfection cells were lysed and reporter gene assays performed. (B) Schematic of a NF-κB1 motif (green) in the mapped region of the PRKD1 promoter and mutational alterations performed to destruct the motif (red). (C,D) Comparison of response of wildtype and mutant PRKD1 promoter luciferase reporter to p65 or KrasG12V. Cells were transfected with vector control or p65 (C) or vector control and KrasG12V, as well as full-length wildtype or mutant versions of the PRKD1 promoter luciferase reporter and renilla-luciferase reporter, as indicated. 24 hours after transfection cells were lysed and reporter gene assays performed. In addition lysates were analyzed by Western blot for expression of p65 (anti-p65) or KRasG12V (anti-FLAG), as well as for β-actin (anti-β-actin). (E) Panc1 cells were transfected with control-shRNA or shRNA targeting expression of KRas, as indicated, for 48 hours. BxPC3 cells were transfected with vector control or KRasG12V as indicated. Chromatin immunoprecipitation (ChIP) was performed using anti-p65 or IgG control and ChIP of p65-bound PRKD1 promoter was detected by PCR. Input controls show PCR for PRKD1 promoter and GAPDH using sheared DNA as a template. (F) Schematic of how oncogenic KRas induces the expression of PKD1 via activation of NF-κB. Red arrows take in consideration published data showing that PKD1 also can activate NF-κB downstream of mutant KRas, this indicating a potential positive feedback loop for signal amplification.
