Fig. 4

KLF10 competed with ELF3 and transcriptionally suppressed Notch-3 and -4. A Primer maps with predicted SP-1/KLF10 binding sites (left panel, predicted p < 0.001) and various designed deletion (right panel) of promoter regions of Notch-3 and -4 for luciferase reporter assay. B Luciferase activity of Panc-1 cells transfected with vector control (yellow), Notch-3(blue), and Notch-4(green) gene promoter plasmids without (filled) or with (diagonal stripe) hemagglutinin (HA) tagged-KLF10 transfection (OE). The bar graphs were mean ± SE from cumulated data of three independent experiments. *p < 0.05 and **p < 0.01, respectively. C Luciferase activity of Panc-1 cells, transfected with Notch-3 (yellow) or Notch-4 (blue) gene promoter plasmids with full length (filled) or with various deletion (diagonal stripe, diagonal check) indicated. The bar graphs were mean ± SE from cumulated data of three independent experiments. *p < 0.05 and **p < 0.01, respectively. D (Upper panel) primer maps of Notch-3/4 gene promoters for CHIP-PCR assay. (Lower panel) CHIP assay. DNA fragments of Panc-1 cells were immune-precipitated with KLF10, IgG or positive control (P) followed by PCR amplification of the Notch-3/4 gene promoter region that contains KLF10 binding sites (input), as described in “Materials and methods” section. E Quantitative PCR of the Notch-3/4 gene promoter region using samples from MiaPaCa (yellow) or Panc-1 (blue) cells prepared as described in D. The bar graphs were mean ± SE from cumulated data of three independent experiments. F MiaPaCa cells with conditional over-expression of KLF10 under Dox treatment were immuno-precipitated with KLF10 antibody and quantitative PCR of the Notch-3 (yellow) and Notch-4 (blue) gene promoter regions. The bar graphs were mean ± SE from cumulated data of three independent experiments. *p < 0.05. G Primer maps of predicted ELF3 binding sites on the promoter regions of Notch-3 (upper panel) and Notch-4 (left panel) genes. H Luciferase reporter assays of Notch-3 (left panel) and Notch-4 (right panel) under conditions of various combination of HA-KLF10 and ELF3 (yellow) and ELF3-mutant (ELF3-S68A, blue) as indicated. The bar graphs were mean ± SE from cumulated data of three independent experiments. **p < 0.01