Sustained DRAQ5 positivity, decreased hemoglobinization, and altered cytomorphology of maturing EpoR-HM erythroblasts. (A) Bone marrow–derived erythroid progenitor cells were expanded in SP34-EX medium and subsequently were cultured in differentiation medium for 40 hours. Frequencies of DRAQ5^negTer119^pos erythroblasts then were determined. (B) In parallel, cultures were analyzed for hemoglobinization (benzidine-positive colonies). (C) Hemoglobin levels in maturing wt-EpoR, EpoR-HM, and EpoR-H erythroblasts were also assayed using diaminofluorene (DAF) and by visualization of pelleted cells. For EpoR-HM erythroblasts, apparently immature morphologies were observed in cytospin preparations (right panel). Micrograph images were visualized using an Olympus IX70 microscope equipped with a 100×/1.3 oil-immersion objective lens (Olympus, Melville, NY). Immersion oil no. 16212 from Cargille Labs (Cedar Grove, NJ) was used with the lens. A Zeiss Axiocam 412312 camera and Axio-vision 4.1 software (Zeiss, Thornwood, NY) were used to capture and process images. In dotplots, the numbers in quadrants (clockwise from bottom left) indicate the percentage of Ter119^negDraq5^neg, Ter119^highDraq5^neg, Ter119^highDraq5^high, and Ter119^negDraq5^pos cells among total live-gated cells. In the bar graphs, plotted values are mean values plus or minus SE; n = 4 wt-EpoR, EpoR-HM, and EpoR-H mice.