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. Author manuscript; available in PMC: 2011 May 10.
Published in final edited form as: Nat Med. 2010 Jul 8;16(8):903–908. doi: 10.1038/nm.2187

Figure 2.

Figure 2

Ectopic MSI2 expression compromises HSC function. Bone marrow from control rtTA (C) or MSI2-transgenic (M) mice was transplanted into irradiated CD45.1+ congenic mice. Six weeks after transplantation, doxycycline was given for 5 d, and bone marrow was analyzed for HSC and progenitor cells. (a) Representative flow cytometric plots, with percentages of cells expressing markers as indicated. The analyzed populations are indicated above the plots. (b) Quantification of long-term HSCs (SLAM: LSK CD150+CD48+ or LSK CD34−). (c) Cell cycle analysis performed with Hoechst and pyronin Y, gated on hematopoietic stem and progenitor cells (LSK cells) (representative experiment from at least seven mice; error bars, s.e.m.). (d) Gene set enrichment analysis (GSEA) on microarray data of MSI2-overexpressing LSKs (Supplementary Methods). (e) Determination of ERK phosphorylation in LSKs after stimulation with SCF. Representative plot shown; histogram indicates combined analysis of five mice from two independent experiments. (f) Determination of symmetric versus asymmetric cell divisions in purified LSKs. Immunofluorescence images stained for Numb (left) and DAPI (right) and bright field (middle). Scale bars, 10 μm. A quantification is presented as pie charts below the images (C, n = 4 experiments, 70% symmetric ± 9.2% (n = 58 cells) and M, n = 5 experiments, 33% symmetric ± 13.2% (n = 61 cells), P < 0.004). (g) White blood cell counts (WBC) in recipient mice administered doxycycline for the indicated lengths of time. Each point represents one mouse; the line indicates the mean WBC of the cohort. (h) Analysis of relative engraftment in peripheral blood after competitive transplants in which MSI2-transgenic (M), or control rtTA (C), bone marrow (CD45.2+) was mixed with competitor wild-type marrow (CD45.1+) at the indicated ratios. (i) Percentage CD45.2+ cells in the long-term HSC (LSK CD34−), LSK and LK cell compartments in the bone marrow of mice transplanted with a 3:1 ratio of MSI2- or rtTA-expressing (CD45.2+) cells to wild-type cells (CD45.1+) at 20 weeks after transplantation, +P < 0.05, **P < 0.001.