No further signaling through the EPO receptor in MPN patients. (A) Representative histogram plots of phospho-STAT5 signaling in control and PV CD34⁻ cells stimulated with 1 IU/mL of EPO (filled histogram) or unstimulated (middle shaded histogram). Dotted line is the isotype control. The relative values are shown in the bar graph (right panel) as the -fold stimulation, which was calculated by dividing the MFI of stimulated cells by that of unstimulated cells. The bar graph demonstrates that CD34⁻ cells from controls demonstrate EPO stimulation. In contrast, PV, ET, and MF patients show no additional stimulation upon EPO addition. (B) Representative histogram plots overlay of the same samples in panel A are shown, this time also gated on the GPA⁺ population. The bar graph demonstrates that CD34⁻/GPA⁺cells from controls demonstrate modest EPO stimulation. In this population, MPN patients show no additional stimulation with EPO. Histogram plots of CD34⁻ /GPA⁺cells from control and PV patients treated with pervanadate demonstrate that both samples can be further stimulated (P-STAT5) upon addition of a tyrosine-phosphatase inhibitor. (C) Representative histogram plots of CD34⁻/GPA⁺cells from a control and a PV patient demonstrate further stimulation of ERK signaling upon treatment with PMA.