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Additional Supporting Information may be found in the online version of this article.

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sc-12-0892_sm_SupplData.pdf120KSupplementary Data
sc-12-0892_sm_SupplFigure1.tif663KSupplementary Figure 1: Generation of Pim1-Tx mice bearing human PIM1 driven by vav- hematopoietic regulatory element and SV40 sequence. (A) The construct used for pronuclear injection. The entire coding DNA sequence of human PIM1 was cloned into vav-regulatory elements and SV40 sequences. (B) Human PIM1 mRNA expression. Human PIM1 mRNA expression was expressed in all hematopoietic tissue but not in non-hematopoietic tissue. Spleen, thymus, bone marrow, and kidney tissues were harvested from WT control mice and Pim1-Tx mice. Messenger RNA was prepared and qRT-PCR performed. The samples were then run on 2% agarose gel. (C) Pim kinase protein expression. Pim1 was over-expressed in the splenocytes of Pim1-Tx mice. Cell lysates were prepared from the splenocytes of Pim1-Tx and WT littermate and were subject to immunoblot analysis using 19F7 Pim1 antibody, which recognizes both human- and mouse-origin of Pim1.
sc-12-0892_sm_SupplFigure2.tif622KSupplementary Figure 2: Pim1-Tx mice have splenomegaly and increased number of HSPCs in the spleen. (A) Increased spleen weight and size in Pim1-Tx mice. Pim1-Tx mice and WT littermates (1.5 yrs old) were sacrificed and spleen was harvested and weighed. Left panel: three representative mice per group were shown (scale bar indicates 1cm). Right panel: average spleen weight in Pim1-Tx mice and WT littermates (n=11, *p<0.05). (B) Increased LSK HSPCs in spleen in Pim1-Tx mice. Red-blood cell- depleted splenocytes from Pim1-Tx mice and WT littermates were stained with Lineage-specific, Scal-1, and c-Kit antibodies and analyzed on flow cytometry. Total number of LSK HSPCs (calculated by total BM mononuclear cells × %LSK) was shown (n=12, *p < 0.05).
sc-12-0892_sm_SupplFigure3.tif1593KSupplementary Figure 3: Pim1-Tx mice show no differences in blood cell subsets and have no evidences of myelodysplastic/myeloproliferative disorder. (A) Peripheral blood cell subset analysis. Peripheral blood cells were stained with Gr-1, B220, and CD3 antibody. The absolute numbers of these cell subsets were calculated as described in the Materials and Methods (n=8). (B) Bone marrow morphology. Bone were harvested from Pim1-Tx mice or wild-type control mice, fixed, sectioned, stained with H/E and examined under microscope (representative of 5 mice in each group, scale bar indicates 20μm). Pim1-Tx mice showed no evidences of myelodysplastic/myeloproliferative disorder.
sc-12-0892_sm_SupplFigure4.tif963KSupplementary Figure 4: Pim1-Tx mice show competitive repopulating units (CRU) comparable to WT littermates at 4 months post transplant. (A) CRUs measured at 6 weeks post transplant. (B) CRUs measured at 4 months post transplant. Various doses of male LSK+CD34- HSCs (15, 45, and 150 cells/recipient mouse) were mixed with 1.5×105 WT female FVB BM cells and transplanted into lethally irradiated female FVB mice. Male donor engraftment was estimated as described at 6 weeks (A: peripheral blood samples; n=29 for WT and n=27 for Pim1-Tx) and 4 months (B: BM samples; n=29 for WT and n=24 for Pim1-Tx) post transplant. Poisson statistical analysis of limitingdilution was performed to allow for estimation of CRUs in each group. The plot shows the percentage of recipient mice containing less than 1% donor male cells at 6 weeks and at 4 months post transplantation versus the number of cells injected per mouse.
sc-12-0892_sm_SupplFigure5.tif854KSupplementary Figure 5: Pim1−/− mice have significantly reduced competitive repopulating units. Limiting-dilution assay was performed as described using Pim1−/− and WT mice. Panels A-C are blood samples collected at 6 weeks post transplant (A: scatter plot; B: CRU frequencies; C: limiting dilution plot). Panel D is BM samples collected at 4 months post transplant and shown the CRU frequency in Pim1−/− and WT mice.
sc-12-0892_sm_SupplFigure6.tif1824KSupplementary Figure 6: Gating strategy to analyze BrdU incorporation in LT-HSCs. BM Lin- cells were labeled with fluorescent-labeled antibodies and analyzed by flow cytometry for the longterm HSCs (LT-HSCs, Lin-Sca-1+c-Kit+CD34-CD135-), Short-term HSCs (ST-HSCs, Lin-Sca-1+c- Kit+CD34+CD135-) and multipotent progenitors (MPPs, Lin-Sca-1+c-Kit+CD34+CD135+). BrdU incorporation was analyzed for LT-HSCs.
sc-12-0892_sm_SupplFigure7.tif384KSupplementary Figure 7: Pim1 regulates LSK cell expansion in vitro. Sorted LSK cells (6,000/well) from WT, Pim1-Tx and Pim1−/− mice were cultured in StemSpan media supplemented with SCF, TPO-1 and Flt3 (100 ng/ml for each) for 4 days. Total cell numbers were counted and averaged from 3-4 wells/group (data are representative of 4 independent experiments).
sc-12-0892_sm_SupplFigure8.tif594KSupplementary Figure 8: Pim1-Tx LSK cells show a reduced rate of apoptosis. BM Lin- cells were enriched by Lineage cell depletion Kit (Miltenyi Biotec) from WT, Pim1-Tx and Pim1−/− mice. The cells were then stained with biotin-labeled lineage specific antibodies, PE-conjugated Scal-1, and APC-H7-conjugated c-Kit followed by streptavidin-Brilliant-violet-605 and Aqua Live/Dead dye. The cells were then fixed and subsequently stained with PE-Cy7-conjugated Ki-67 antibody and FITC- conjugated Caspase 3 antibody. All the cells were gated on LSK population.
sc-12-0892_sm_SupplFigure9.tif840KSupplementary Figure 9: Cytokines/growth factors up-regulate the expression of Pim1, but not Pim2 or Pim3. BM Lin- cells isolated from C56B/6 mice were cultured in DMEM medium supplemented with 20% heat-inactive FBS and 100 ng/ml of SCF, IL-3, IL-6, TPO, Flt3 alone or in combination for 24 hrs. Cells were harvested and mRNA isolated for RT-PCR analysis of Pim1 (A), Pim2 (B), and Pim3 (C) gene expression.

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