Fig. 1

Pim1 regulates c-Kit expression in murine HSPCs. a c-Kit expression is down-regulated in HSPCs from Pim1^−/− and Pim TKO mice. Bone marrow cells were harvested from age-matched WT, Pim1^−/−, Pim2^−/−, Pim3^−/−, Pim TKO, or Pim1 transgenic (Pim1-Tx) mice. Lineage negative (Lin⁻) cells were enriched and stained with PE-conjugated c-Kit antibody. The expression of c-Kit was analyzed by flow cytometry. FACS contour plots were shown. Data represent 3 independent experiments (>3 mice per group in each experiment). b c-Kit expression is down-regulated in SP cells from Pim1^−/− and Pim TKO mice. BM Lin^- cells obtained from WT, Pim1^−/−, Pim2^−/− and Pim TKO mice were stained with Hoechst 33342 followed by c-Kit antibody surface staining. The histograms of c-Kit expression on side population (SP) cells were shown. Data represent 2 independent experiments. c The expression of Sca-1 and Flt3 was not affected in HSPCs from Pim1^−/− and Pim TKO mice. Top panel: BM Lin⁻ cells were harvested from WT, Pim1^−/−, Pim2^−/−, or Pim TKO mice and stained with PE-conjugated Sca-1 antibody. FACS contour plots of Scal-1 were shown. Bottom panel: BM Lin⁻ cells were harvested from WT, Pim1^−/−, Pim2^−/−, or Pim TKO mice and stained with Flt3 (CD135) antibody. Histogram overlay plots of Flt3 were shown. Data represent 3 independent experiments. d The c-Kit receptor signaling in response to SCF stimulation was reduced in HSPCs from Pim TKO mice. BM Lin^- cells obtained from WT or Pim TKO mice were stimulated with SCF (final concentration of 100 ng/ml) for 10 min and then co-stained with Scal-1 antibody, c-Kit antibody, and p-ERK/p-STAT3 antibody as described in “Methods and materials” section. The level of p-ERK or p-STAT3 was gated on LSK cells. MFI mean fluorescent intensity. Data represent 2 independent experiments