FIG. 10.

B19V infection of CD36⁺/Epo⁺ EPCs is influenced by the Epo concentration in the culture medium. Day 4 stocks of CD34⁺ HSCs expanded in Wong medium were cultured in Wong medium containing Epo at the indicated concentrations until day 8, when B19V infection was carried out. Infected cells were analyzed at 48 h p.i. (A) Epo pathway signaling in infected cells was assessed by flow cytometry for p-EpoR, pJak2, and B19V NS1 (left) and quantification using MFI for pEpoR and pJak2 staining (right). (B) RT-qPCR-based mRNA quantification for VP2-encoding and D1/A1-2-spliced mRNAs, normalized to the level of β-actin mRNA. (C) Virus entry and genome replication. B19V genome copy numbers are expressed as gc/cell. Levels of virus entry were determined as described in Materials and Methods, and fold differences between replication and virus entry are shown. Averages and standard deviations are shown in panels B and C and were calculated from at least three independent experiments. (D) Cell viability and cell cycle stage in CD36⁺/Epo⁺ EPCs expanded in Wong medium containing Epo at a concentration of 0.1, 0.5, 2, or 10 units/ml, as indicated, from days 4 through 8. Cells were then analyzed for cell death by annexin V/PI staining and cell cycle stage by DAPI staining. For annexin V/PI staining, the numbers shown in the lower left quadrants represent the percentages of live cells, and the numbers in upper and lower right quadrants represent the percentages of later/early apoptotic cells. In the case of DAPI staining, the numbers represent the percentages of cells in each phase of the cell cycle (G₀/G₁, S, and G₂/M).