Fig 2: Pim1 and Notch1 synergize to promote breast and prostate xenograft growthThe 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) N1?E 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. ß-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).

Fig 3: Effect of miR-1236-3p on the expression Pim-3, p-Bad and Bcl-xL in cultured neurons. The protein levels of Pim-3, p-Bad (Ser112), Bcl-xL in cultured neurons were detected via western blot analysis. miR, microRNA; p, phosphorylated; siRNA, small interfering RNA.

Fig 4: Overexpression of PIM3 in hepatoblastoma cells results in increased stem cell-like cancer cell (SCLCC) phenotype and decreased cisplatin sensitivity. HuH6 cells were stably transfected with a pcDNA3.1/V5-HisC-based expression vector for PIM3 overexpression (PIM3 OE) or an empty vector (EV) control. (A) Immunoblotting of whole cell lysates verified PIM3 overexpression. ß-actin was used to confirm equal protein loading. (B) CD133 cell surface expression was evaluated by FACS in HuH6 EV and PIM3 OE cells and reported as percent CD133 expression ± SEM. CD133 expression was increased significantly in the PIM3 OE cells. Representative contour plots are shown to the right of the graph. (C) Quantitative real-time PCR was utilized to assess the mRNA abundance of Oct4, Nanog, and Sox2 in HuH6 EV and PIM3 OE cells. Relative abundance of mRNA was calculated using the ??Ct method and reported as mean ± SEM of three biologic replicate experiments. PIM3 OE cells had significantly increased abundance of all three stem cell markers compared to HuH6 EV cells (p < 0.001), further indicating an increase in the SCLCC phenotype following overexpression of PIM3. (D) The effect of PIM3 OE on hepatoblastoma cell viability following treatment with cisplatin was evaluated with alamarBlue assay. HUH6 EV and PIM3 OE cells were treated with increasing concentrations of cisplatin (0–100 µM) for 72 h and viability measured. PIM3 OE cells were significantly less sensitive to cisplatin than HuH6 EV cells, indicating that PIM3 contributes to cisplatin insensitivity.

Fig 5: PIM kinases were expressed in HuH6 cells, the COA67 PDX, the parent tumor for COA67, and human hepatoblastoma tumor specimens(A) Immunoblotting for PIM1, PIM2, and PIM3 was completed on HuH6 and COA67 cell lysates with ß-actin controls. All three PIM proteins were present in both cell lines. (B) Immunohistochemistry for PIM3 was performed on human hepatoblastoma (top left panel, magnification on bottom left panel) and normal liver (top right panel) with negative staining in the IgG control is in bottom right insert, not bottom right panel as currently stated. PIM3 was more highly expressed in hepatoblastoma (top left panel) than normal liver (top right panel). Black arrow in bottom left panel indicates lack of staining in the peri-portal connective tissue in comparison to the PIM3 stained hepatoblastoma cells in the left half of the photomicrograph. (C) Hematoxylin and eosin (top panel) and immunohistochemistry for PIM3 (bottom panel) with IgG control (inserts, bottom panels) in parent tumor (left bottom panel) and COA67 PDX (right bottom panel) revealed that the COA67 PDX closely resembled the parent tumor histomorphologically and PIM3 was present in both the PDX and its parent.