EpoR-HM is attenuated in its ability to support Epo concentration-dependent 3HdT incorporation in bone marrow–derived EPCs. (A) Marrow cell preparations were cultured in HCEM in the presence (or absence) of either Epo (0.2 U/mL) or IL-3 (10 ng/mL). At the indicated intervals, rates of cytokine-dependent proliferation were assayed based on stimulated rates of 3HdT incorporation. (mean incorporation rates ± SD, n = 3). (B) Diagrammed are EpoR-HM and wt-EpoR forms together with box1-associated JAK2 kinase and cytoplasmic phosphotyrosine sites within the wt-EpoR. (C) Marrow preparations from EpoR-HM or wt-EpoR control mice were cultured in the presence of Epo at the indexed concentrations. At 14 hours of culture, rates of Epo-dependent 3HdT incorporation were determined. Mean incorporation values (± SD) for 3 wt-EpoR and 3 EpoR-HM mice are shown. (D) To confirm apparent differences in Epo-dependent 3HdT incorporation as supported by EpoR-HM versus wt-EpoR, EPCs from EpoR-HM and control mice also were expended in the presence of dexamethasone, SCF, and Epo for 65 hours and were then tested for Epo responsiveness. Values are means ± SD of triplicate samples for 2 wt-EpoR and EpoR-HM mice (and outcomes are representative of 2 independent repeated experiments).
