EPO/EPOR signaling in SS erythroid cells was impaired. (A-B) Representative western blot analysis of phosphorylated Stat5 (pStat5) in the enriched BM erythroid cells of AA and SS mice and its quantification. The fold changes were normalized to the pStat5 level in BM erythroid cells of AA mice stimulated with EPO at 1 U/mL. (C-D) Representative flow-cytometric analysis of pStat5 in erythroid cells at each developmental stage and its quantification. (E-F) Representative western blot analysis of Jak2 and phosphorylated Jak2 (pJak2) in the enriched BM erythroid cells of AA and SS mice and its quantification (N = 3). (G) Representative western blot analysis of Erk and phosphorylated Erk (pErk) in the enriched BM erythroid cells of AA and SS mice. (H) Quantification analysis showing the fold change of Erk protein level. Actin was used as reference protein. (I) Quantification analysis of pErk in the enriched BM erythroid cells of AA and SS mice. The fold changes were normalized to the pErk level (normalized with Erk level) in enriched BM erythroid cells of AA mice stimulated with EPO at 1 U/mL. The quantitative analyses of pStat5 and pErk were based on data from 3 biological replicates (N = 3). (J) and (K) Representative western blot and quantitative analyses of EpoR levels in the enriched BM erythroid cells from AA and SS mice (N = 3). (L) Fold change of BM erythroblast numbers in 2 femurs + 2 tibias, and (M) fold change of CFU-E colony numbers in 5 × 104 BM cells of PBS- or EPO-injected AA and SS mice (N = 6). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Poly, polychromatic erythroblast.
