Oncostatin-M promotes the survival of Kit^–CD71^high erythroblasts, in part via EpoR PY343–dependent routes. (A) Oncostatin-M reinforcement of EpoR-H, but not EpoR-HM, erythroblast survival. EpoR-H and EpoR-HM erythroblasts were expanded in SP34-EX in Epo (2.5 U/ml) and SCF (5 ng/ml) in the presence or absence of oncostatin-M (onco-M) at 10 ng/ml. At 72 hours of culture, frequencies of annexin V–positive cells among Kit^–CD71^high and Kit⁺CD71^high erythroblasts were determined. Note the substantial survival effect of oncostatin-M selectively on EpoR-H Kit^–CD71^high erythroblasts (upper left panels) (*P < 0.01 vs. EpoR-H with oncostatin-M; ^#P < 0.05 vs. EpoR-HM with oncostatin-M). Also illustrated are annexin V staining profiles for expanded EpoR-H and EpoR-HM cells, and for CD71^high erythroblasts. For expanded erythroblasts from EpoR-H and EpoR-HM bone marrow preparations, Kit⁺CD71^low, Kit⁺CD71^high, and Kit^–CD71^high subpopulations also were isolated by FACS, and lysed in TRIzol. Levels of oncostatin-M receptor β (onco-MRβ) (and, for comparison, oncostatin-M transcripts) from each population then were determined by quantitative RT-PCR. Actin was used as a normalizing control. (B) Oncostatin-M selectively activates Stat3 and Stat1 in CD71^high erythroblasts. WT-EpoR CD71^high erythroblasts were isolated from expansion cultures. Erythroblasts then were deprived of cytokines for 6 hours and exposed to Epo (2.5 U/ml) or oncostatin-M (10 ng/ml). At 10 minutes of exposure, lysates were prepared, and levels of activated (PY) and total Stat3, Stat1, and Stat5 were analyzed by Western blotting. (C) A model is outlined in which EpoR-PY343 (and Stat5) signals act during stress erythropoiesis to centrally support erythroblast production, reinforce Kit signals for proerythroblast expansion, and mediate EpoR plus oncostatin-M signals for late erythroblast survival.