JNK is activated and promotes proliferation in human EPO-responsive leukemia cells. (A) Western blot analysis of HCD57 (lanes 1 and 2), TF-1 (lanes 3 and 4), and UT7-EPO (lanes 5 and 6) deprived of EPO for 4 hours (lanes 1, 3, and 5) and then stimulated with EPO for 5 minutes (lanes 2, 4, and 6). Phosphorylation of JNK1 (p-JNK1) and JNK2 (p-JNK2) was assessed by immunoblotting with anti–phospho-JNK antibody. ERK2 phosphorylation that cross-reacts with the phospho-JNK antibody is also detected (p-ERK2). (B) Effect of SP600125 inhibitor on proliferation by MTT assay (left panels) and apoptosis by flow cytometry of propidium iodide–stained cells (right panels) of UT7-EPO (top panels) and TF-1 (bottom panels) cells. UT7-EPO and TF-1 cells were cultured in EPO alone (–), EPO plus DMSO vehicle (Vehicle), or EPO + 12.5 μM and 25 μM SP600125 for 48 hours. Treatment with 12.5 μM SP600125 inhibited proliferation by approximately 60% in both UT7-EPO and TF-1 cells, whereas the apoptosis observed was less than 10%.