Hemin injection led to suppression of BM erythropoiesis and inhibition of EPO/EPOR signaling in erythroid cells via an indirect mechanism. (A) Effects of hemin injection on BM BFU-E colonies, CFU-E colonies, and erythroblasts in AA mice (N = 6). (B) Quantitative analysis of pStat5 levels as assessed by flow cytometry in BM erythroid cells of PBS- or hemin-injected AA mice. (C-D) Representative western blot analysis of pErk in the enriched BM erythroid cells from PBS- or hemin-injected AA mice and its quantification. The fold changes of pErk were normalized to the pErk level in BM erythroid cells of PBS-injected AA mice stimulated with EPO at 1 U/mL. (E) Effects of RBC lysate injection on BM BFU-E colonies, CFU-E colonies, and erythroblasts in WT B6 mice (N = 6). (F) Quantitative analysis of pStat5 levels as assessed by flow cytometry in BM erythroid cells of PBS- or RBC lysate–injected WT B6 mice. (G-H) Representative western blot analysis of pErk level in enriched BM erythroid cells of WT B6 mice injected with PBS or RBC lysate and its quantification. The fold changes of pErk were normalized to the pErk level in BM erythroid cells of PBS-injected WT B6 mice stimulated with EPO at 1 U/mL. The quantitative analysis of pStat5 and pErk were based on data from 3 biological replicates (N = 3). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.