EPO alone sustains cell survival in ETV6-RUNX1–expressing cells. (A) Growth curves: A total of 500 000 uninduced and ETV6-RUNX1–expressing BaF3 1/27 cells were cultured in the absence of IL-3 and absence or presence of 20 U/mL human EPO for up to 8 days (n = 4 P = .0085). Only in ETV6-RUNX1–expressing cells was EPO able to maintain cell survival in the absence of IL-3 but not in the absence of EPOR (siRNA knockdown) or intact JAK2 activity (AG490). (B) Apoptosis analysis: On day 8, cells were stained with propidium iodide, and the sub G1 population (apoptotic cells) was analyzed. In the long-term assay, only cells expressing ETV6-RUNX1 maintained low levels of apoptosis. (C) Growth competition experiment: A total of 105 cells of mifepristone-induced clone 1 control cells and 1/27 cells expressing ETV6-RUNX1 were mixed and grown for 4 days in the absence of IL-3 and the presence of 20 U/mL EPO. Cells were stained at day 0 and day 4 with α-V5-FITC antibody and 4,6-diamidino-2-phenylindole, and a minimum of 200 cells were counted for ETV6-RUNX1 expression. Images were taken with an Axioscope microscope (Zeiss) with 63×/1.25 oil and 100×/1.30 oil objectives in a Vecta shield mounting medium H-1000 (Vector Laboratories Inc). A Hamamatsu digital camera ORLA-ER (C-4747-80) was used and images were captured with smart Capture X Version 2.6.2 and Adobe Photoshop CS Version 8.0. (D) Analysis of cell cycle: On day 4 of growth in the absence or presence of IL-3 or EPO, cells were stained with Hoechst 33342 and pyronin Y (n = 3; P = .0095). Only in ETV6-RUNX1–expressing cells could EPO induce G1 arrest of cell cycle, but not quiescence, measured as a percentage of cells in G0 (see also supplemental Figure 5).
