Fig 2: Phosphorylation of RBMY by its upstream kinases. (A) Hierarchical clustering of RBMY-bound protein kinases, which were shown by analysis of a human protein array, based on Z-score classification. (B) An in vitro kinase assay followed by immunoblotting showed the phosphorylation of His-RBMY (His-RBMY-p) by the indicated kinases. Phosphorylation reactions were performed in the presence or absence of ATP. (C) Co-IP–WB on Huh-7 cells overexpressed with GFP-RBMY and HA-kinases are indicated. Ten percent input of total protein cell lysate used for co-IP–WB is shown. The blot showed clear bands of different phosphorylation patterns of RBMY by its upstream kinases. Blotting of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) verifies equal protein input. GAPDH was absent in the immunocomplex, confirming the specificity and purity of anti-GFP immunoprecipitation. (D) CIP dephosphorylated His-RBMY on Ser/Thr residues. Recombinant His-RBMY proteins were phosphorylated by His-PIM1 and His-CK1e in an in vitro kinase assay. The phosphorylated His-RBMY proteins were purified using His-tagged protein purification spin columns, and the eluted proteins were subjected to CIP treatment and analyzed by Western blot using anti-RBMY antibody (SRGY clone 1). (E) Dephosphorylation of GFP-RBMY by CIP. Huh-7 cells cotransfected with GFP-RBMY and HA-kinases were harvested for CIP (20 U) treatment. The result was revealed by immunoblotting using SRGY clone 1 antibody. (F) Immunoprecipitation assay targeting the endogenous RBMY was performed on Huh-7 cells transfected with or without HA-PIM1. Anti-RBMY antibody (SRGY clone 1) was used to precipitate the phosphorylated RBMY (RBMY-p) mediated by HA-PIM1 (lane 5) and other endogenous kinases (lane 6). CIP treatment then was performed to dephosphorylate the immunoprecipitated RBMY-p. Western blot was accomplished with SRGY clone 1 monoclonal antibody, which recognized RBMY-p (lanes 5 and 6) and the nonphosphorylated RBMY (lanes 7 and 8). (G) Schematic illustration of domain structures of RBMY deletion mutants. These mutants bound (+) or did not bind (-) PIM1 p34 as indicated. Co-IP–WB using anti-HA and anti-GFP antibodies identified the RBMY domain essential for PIM1 interaction. Detection of GAPDH confirms equal protein input and the specificity of anti-HA and anti-GFP immunoprecipitation. The specific bands of interest are indicated by red arrowheads and the asterisk shows the nonspecific bands yielded by using anti-HA antibody for immunoprecipitation. Ab, antibody.

Fig 3: Pim kinases enhance endogenous Notch activityA. Endogenous expression levels for intracellular domains of Notch1 (N1ICD) or Notch3 (N3ICD), and for Pim family kinases were analysed by Western blotting from MCF-7 and PC-3 cell lysates. ß-actin levels were used as loading controls. B. Endogenous Notch activity was measured by CSL-dependent luciferase reporter assays in MCF-7 transiently transfected with non-targeting (NT) or Pim gene-specific siRNAs. RLU, relative light unit. C. Similar assays were carried out in cells transiently transfected with an empty control vector (C), wild-type (WT) or kinase-deficient (KD) Pim1. D-G. CSL- or CMV-dependent luciferase reporter assays were performed with untransfected MCF-7 or PC-3 cells that had been treated for 24 h with 0.1% DMSO (-), 10 µM DHPCC-9 or 10 µM SGI-1776. Shown are representative graphs from three independent luciferase experiments with average data from three parallel samples.

Fig 4: 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 5: Phosphorylation of Notch1 increases PC-3 cell migrationA. PC-3 cell migration was analysed by wound healing assays in cells transiently transfected with Pim1 and/or treated with 0.1% DMSO, 10 µM DHPCC-9 or 5 µg/ml of DAPT for 24 h. Pim expression levels from the wound healing samples were analysed by Western blotting. B. Similar wound healing assays were performed with PC-3 cells that had been plated onto wells coated with the Notch ligand Jagged1. C. N1?E wild-type (WT), phosphodeficient (SA) or phosphomimicking (SE) mutants were overexpressed in PC-3 cells, in which cell migration was analysed after treatments with 0.1% DMSO or 10 µM DHPCC-9. Shown are average healing percentages from two or three independent experiments.