Association of functional EPOR expression with ETV6-RUNX1⁺ leukemic cell lines and patient samples. (A-B) Western blot analysis of EPOR expression using Amgen antibody A82 in different ALL cell lines and CD19⁺ cells isolated from patients diagnosed with ALL. K562 and HeLa cell lines were used as controls for low and negative expression of EPOR, respectively, and HEL cells for high expression. As a direct comparison with REH cells, the SEM, Nalm6, and Raji cell lines were used as pre-B cell lymphoid ETV6-RUNX1-negative cell lines. ETV6-RUNX1-positive and -negative patients are designated ER⁺ and ER⁻, respectively. (C) Functional EPOR is ectopically expressed on the cell surface of CD19⁺ cells isolated from ETV6-RUNX1⁺ pre-B ALL patients (ER⁺) and leukemic cell lines but not ETV6-RUNX1-negative cases (ER⁻). Washed cell lines and CD19⁺ sorted patient cells were incubated with biotinylated EPO that binds to the EPOR. Avidin-fluorescein was added and the amount of EPO measured as the increase in fluorescence analyzed by FACS compared with controls (see also supplemental Figure 1).