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. 2014 Feb 10;211(2):181–188. doi: 10.1084/jem.20131189

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© 2014 Grover et al.

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Figure 1. — Systemic Epo treatment introduces E bias in myelo-erythroid progenitor and LMPP production. (A) In vitro E differentiation potential of 500 bone marrow Lin⁻Sca-1⁻c-Kit⁺ cells isolated after 2 d of Epo treatment (Epo) or mock treatment (control), measured by 2,7-diaminofluorene (DAF) staining of M3434 methylcellulose cultures after 8 d of culture. Values are mean ± SD, n = 3. One of two representative experiments is shown. (B) Total colonies formed from cells plated in A. (C) Colony-forming potential of 2-d Epo-exposed or control Lin⁻Sca-1⁻c-Kit⁺ bone marrow cells was assayed separately under GM (M3534; 500 cells), E (M3436; 1,000 cells), preB (M3630; 1,000 cells), and Mk (MegaCult; 500 cells) conditions. Values are mean ± SD, n = 3. One of two representative experiments is shown. (D) Bar graph showing serum cytokine levels in mice hydrodynamically injected with pCMV6-Epo or empty pCMV6 vector 2 d after transfection. Values are mean ± SD, n = 8, from two experiments. (E) Bar graphs showing total bone marrow cellularity (femurs and tibiae) in mice hydrodynamically injected with pCMV6-Epo vector (Epo; n = 6) or pCMV6 vector (control; n = 5), as indicated. Analysis was performed 2 d after injection. Values are mean ± SD, n = 5 (control) and 6 (Epo). One of three representative experiments is shown. (F) Bar graphs showing Lin⁻Sca1⁻c-Kit⁺IL7rα⁻ myelo-erythroid progenitor cells from mice in E as percentage of live singlets. (G) Representative flow cytometric analysis of the hematopoietic myelo-erythroid progenitor population from bone marrow of wild-type C57BL/6 mice after 2 d of Epo exposure. The size of gated populations as percentage of the parental population is shown next to each gate. (H) Bar graphs representing the mean size of each myelo-erythroid progenitor population as defined in G, shown as percentage of the total Lin/Sca-1/IL7Rα⁻c-Kit⁺ progenitor fraction in 2-d Epo-exposed (Epo) and control mice. Values are mean ± SD, n = 5, from two experiments. (I) Bar graphs represent the frequencies of LSK subpopulations in bone marrow of 2-d Epo-exposed and control mice. Values are mean ± SD, n = 4. One of two representative experiments is shown. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.