Epo-dependent macrophage-derived PGE₂ signaling enhances SEP differentiation. (A) LC-MS/MS analysis of extracellular PGE₂ (left y-axis) and mRNA expression of PTGES (right y-axis) in mouse BMDMs at the indicated time points before and after Epo treatment. One-way ANOVA followed by Dunnett’s multiple comparisons. (*) Represents P values for comparisons between indicated time points and 0 time point; (red *), PTGES; (black *), PGE₂ (n = 3 per time point). (B) ELISA analysis of serum Epo and spleen PGE₂ on indicated days after BMT. In all, 500 000 unfractionated bone marrow cells were transplanted to C57BL/6 recipients. For each time point, n = 3 mice per group. (C) LC-MS/MS analysis of extracellular PGE₂ of human BMDMs at the indicated time points before and after Epo treatment. (D-E) Analysis of manipulating PGE₂ signaling in in vitro SEP cultures. (D) SEPs cultured in SEEM were treated with or without 50 nM 16,16-dimethyl PGE₂. Flow cytometry analysis of in vitro cultured SEPs. Percentage of CD34⁺CD133⁺KS SEPs in total cell population (left); total cell numbers after culture (right). (E) SEPs were cultured in SEDM with or without 10 μM CAY10526 (mPGESi, the PTGES inhibitor). Flow cytometry analysis of in vitro cultured SEPs. Percentage of CD34⁺CD133⁺KS SEPs in total cell population (left); total cell numbers after culture (right). (F-G) Bone marrow cells were expanded in SEEM and treated with Epo, PGE₂, mPGESi, or WIF1, as indicated. (H) Schematic of macrophage-SEP interactions and the changes in signaling induced by macrophage EpoR signaling. Student t test (2-tailed). Data represent means ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.