Figure 9. Pim1 and Notch1 synergize to promote breast and prostate xenograft growth.

The chorioallantoic membrane (CAM) model was used to measure tumorigenic growth of xenografted MCF-7 or PC-3 cells. A. MCF-7 cells transiently transfected with wild-type (WT), phosphodeficient (SA) or phosphomimicking (SE) N1ΔE were grown for 5 days on the CAM and treated daily with 30 μl of water-diluted 100 μM estradiol (E2) -/+ 200 μM DHPCC-9. B. Similar experiments were carried out also in the absence of E2. C. Pim expression levels were analysed by Western blotting from MCF-7 cells cultured in the presence of increasing concentrations of estradiol. β-actin staining was used as a loading control. D. Untransfected PC-3 cells were grown on the CAM for 5 days and treated daily with DMSO, 100-200 μM DHPCC-9 or 5 μg/ml of DAPT. A minimum of three separate experiments were performed. Shown are average tumor sizes from one representative experiment. E. N1ICD protein was stained from paraffin-embedded samples of orthotopic prostate xenografts that had been formed by mock-transfected PC-3 cells or cell stably overexpressing Pim1 or Pim3 [25]. Part of the mice with Pim-3-expressing xenografts had been daily treated with 50 mg/kg of DHPCC-9. After scanning, manual double-blinded analyses were performed to non-necrotic tissue sections. Shown are the average numbers of cells with nuclear Noth1 ICD. F. Representative images show cleaved Notch1 staining (brown).