Figure 1
From: The PRKD1 promoter is a target of the KRas-NF-κB pathway in pancreatic cancer
KRas regulates PKD1 expression by activating PRKD1 gene transcription.
(A) Sections of normal mouse pancreas, pancreata from 18 week old LSL-KrasG12D/+;p53R172H/+;Pdx1cre/+(KPC) mice or pancreata from 10 week old p48cre/+;LSL-KrasG12D/+(KC) mice were analyzed by H&E staining and by immunohistochemistry for PKD1 expression. Shown is a characteristic area of the pancreas. (B) Whole cell lysates of indicated PDA cell lines or normal control (HPDE) were analyzed by Western blotting for expression of PKD1 (anti-PKD1). Probing lysates for β-actin (anti-β-actin) served as loading control. (C) Panc1 cells were transfected with control-shRNA or shRNA targeting expression of KRas as indicated. 48 hours after transfection cells were lysed, PKD1 was immunoprecipitated (anti-PKD1), samples separated on SDS-PAGE and analyzed by immunoblotting for PKD1 expression (anti-PKD1). In addition, lysates were analyzed by Western blotting for efficient knockdown (anti-KRas). Probing lysates for β-actin (anti-β-actin) served as a loading control. (D,E) HPDE or BxPC3 cells were transfected with KRasG12V or control vector as indicated. 48 hours after transfection cells were lysed, PKD1 was immunoprecipitated (anti-PKD1 antibody), samples separated on SDS-PAGE and analyzed by immunoblotting for PKD1 expression (anti-PKD1 antibody). In addition, lysates were analyzed by Western blotting for KRas knockdown (anti-KRas) and for β-actin (anti-β-actin) as a loading control. (F) Indicated cell lines were cultivated under normal growth conditions. mRNA was isolated and the expression of PKD1 and β-actin was detected by RT-PCR. (G) BxPC3 cell were transfected with KRasG12V or control vector. 48 hours after transfection a qPCR was performed. Shown is relative fold PKD1 expression normalized to GAPDH. The asterisk indicates statistical significance. (H–J) BxPC3 (H) or HPDE (I) cells were transfected with KRasG12V or vector control, PRKD1-luciferase reporter and renilla-luciferase reporter; Panc1 (J) cells were transfected with control-shRNA or shRNA targeting KRas (two different sequences, #1 and #2), PRKD1-luciferase reporter and renilla-luciferase reporter. 48 hours after transfection cells were lysed and reporter gene assays performed. In addition lysates of H, I were analyzed by Western blot for KRasG12V expression (anti-FLAG), lysates of J for knockdown of KRas (anti-KRas), as well as for β-actin (anti-β-actin) as loading controls.
