Isolation of Lin^–CD34⁺ and Lin^–CD34^– EML cells. (A) Expression of the lineage markers on EML cells before lineage depletion (dotted line) or after lineage depletion (solid line) by magnetic cell sorting. The cells were stained with phycoerythrin (PE)-cy7-lineage mixture antibodies and analyzed by FACS. (B) Lin^–-enriched EML cells were stained with PE-cy5-lineage mixture antibodies, PE-conjugated CD34 antibody, and allophycocyanin-conjugated Sca-1 antibody. Subsequently, the Lin⁺ cells were gated out and Lin^– EML cells were sorted into CD34⁺ and CD34^– EML cell populations by Moflo high flow cell cytometry. (C) Lin^–CD34^– and Lin^–CD34⁺ EML cells cytospun onto slides were stained with Wright-Giemsa by standard protocols. (Magnification: ×400.)

The expression of dGFP driven by the β-globin promoter alone or with an upstream insert of the HS2 enhancer. (A) The map of the integrated β promoter-dGFP (Upper) and the expression of dGFP in Lin^–CD34⁺ or Lin^–CD34^– EML cells infected with a lentiviral reporter containing the β-promoter-dGFP cassette (Lower). PPT, polypurine tract. (B) The map of the integrated HS2-β promoter-dGFP (Upper) and the expression of dGFP in Lin^–CD34⁺ or Lin^–CD34^– EML cells infected with a lentiviral reporter containing the HS2-β-promoter-dGFP cassette (Lower). (C) Seventy five nanograms of genomic DNA from parental EML cells (no lentivirus infection) or Lin^–CD34⁺dGFP⁺ and Lin^–CD34⁺dGFP^– EML cells (infected with a lentiviral reporter containing the cassette of HS2-β-promoter-dGFP) was amplified with the primers for the dGFP gene by PCR. This process showed that there was a similar amount of lentiviral DNA in each of the two cell types, even though GFP expression was lower in the CD34⁺ cells.

The differentiation and proliferation of Lin^–CD34^– or Lin^–CD34⁺EML cells in response to different cytokines. (A) The expression of cell surface markers CD34 and Sca-1 on Lin^–CD34⁺ EML cells in SCF medium at different times, as analyzed by FACS. (a) Purification check after the sorting. (b–d) Lin^–CD34⁺ cells were subcultured in SCF medium after 1 day (b), 2 days (c), and 4 days (d). (B) The growth curve of the sorted Lin^–CD34^– and Lin^–CD34⁺ EML cells incubated in Iscove's modified Dulbecco medium supplemented with 15% horse serum in the presence of SCF (100 ng/ml), IL-3 (5 ng/ml), or a combination of SCF (100 ng/ml) and IL-3 (5 ng/ml) for different times. The live cells were counted by using trypan blue staining. The data represent three independent experiments.

Principal component analysis of transcription factor expression by Lin^–CD34⁺, Lin^–CD34^–, parental EML, and different differentiated EML cells. Principal component analysis allows us to present the distribution of the samples in the multidimensional transcription factor expression space in a 2D graph. A preliminary step of the analysis is a binormalization procedure that simultaneously centers the transcription factor profile of each sample and expression intensities across all samples of each transcription factor around zero. The principal components are linear combinations of the normalized transcription factor expression profiles. The first two leading principal components capture most of the variation of the data. Therefore, the data can be displayed (with a minor loss of information) in a 2D graph by projection of the 19 samples onto a subspace spanned by the two leading principal components. The axes legends pc1 and pc2 stand for the first two principal components. The distances between the samples in this 2D space is an approximation of the dissimilarities between their corresponding transcription factor expression profiles. The cell types are represented by the symbols indicated in Inset.

The differentiation of Lin^–CD34⁺dGFP^low and Lin^–CD34⁺dGFP^– EML cells transfected with HS2–2b-dGFP lentivirus. (A) The expression of dGFP in CD34⁺ or CD34^– EML cells differentiated from Lin^–CD34⁺dGFP^low EML cells. (a) The purification of the sorted Lin^–CD34⁺dGFP^low. (b–d) The sorted Lin^–CD34⁺dGFP^low cells were cultured in SCF medium for 1 day (b), 2 days (c), and 3 days (d). (B) The expression of dGFP in CD34⁺ or CD34^– EML cells differentiated from Lin^–CD34⁺dGFP^– EML cells. (a) The purification of the sorted Lin^–CD34⁺dGFP^–. (b–d) The sorted Lin^–CD34⁺dGFP^– cells were cultured in SCF medium for 1 day (b), 2 days (c), and 3 days (d).

The phosphorylation of c-Kit or IL-3R/β in Lin^–CD34^– and Lin^–CD34⁺EML cells in response to different cytokines. (A) Total cell lysates of Lin^–CD34⁺ and Lin^–CD34^– EML cells were separated by SDS/PAGE and transferred to an Immobion-P membrane. The membrane was probed with anti-c-Kit antibody, stripped, and reprobed with anti-β-tubulin antibody. IB, immunoblotting. (B and C) Lin^–CD34⁺ (B) and Lin^–CD34^– (C) EML cells were starved and treated with no stimulation, SCF (100 ng/ml), IL-3 (5 ng/ml), or the combination IL-3 (5 ng/ml) and SCF (100 ng/ml) for 10 min. Cells were harvested, and c-Kit was immunopreciptated (IP) with c-Kit antibody (M14). The immunopreciptates were analyzed by SDS/PAGE, followed by immunoblotting with the indicated antibodies. After being hybridized with antiphosphotyrosine Y719-specific c-Kit antibody, the same membrane was stripped and reprobed with the general antiphosphotyrosine antibody PY100 and anti-c-Kit antibody. (D) Lin^–CD34^– EML cells were starved and treated with no stimulation, SCF (100 ng/ml), IL-3 (5 ng/ml), or the combination IL-3 (5 ng/ml) and SCF (100 ng/ml) for 10 min. Cells were harvested, and IL-3R/β was immunopreciptated with anti-IL-3R/β antibody (K17). The immunopreciptates was analyzed by SDS/PAGE, followed by immunoblotting with antiphosphotyrosine antibody PY100. The membrane was then striped and reprobed with IL-3R/β antibody.