ERFE overexpression augments ineffective extramedullary erythropoiesis in older adult thalassemic mice. (A-B) Mice generated from Th3 × E(M) breeding were injected retroorbitally with NHS-Sulfo-Biotin 4 times in 48 hours, and then ∼50 μL weekly cheek bleeds were performed to obtain peripheral blood for analysis. Erythroid cells were analyzed via flow cytometry using PE-conjugated–anti-TER119 to identify erythroid lineage, APC-conjugated streptavidin to detect biotinylation, and TO-1 to detect RNA-containing reticulocytes. (A) Percentage of biotinylated erythrocytes over the course of 5 weeks (color scheme same as that in panels B-F). (B) Percent reticulocytes at terminal bleed defined as TER119⁺; TO-1⁺ cells relative to TER119⁺ cells. (C-F) Erythroid cells in BMs and spleens were harvested from mice at ∼37 to 45 weeks. Dead cells, and CD45⁺ cells, were excluded by 7-AAD and CD45 staining, and live erythroid cells from BM (C) or spleen (D) were counted via flow cytometry as live TER119⁺ cells. (E) Spleen weight as the percentage of body weight (F) Total splenic erythroid cells indices adjusted for body size were estimated by multiplying the values in panel D with the relative spleen size in panel E. (B-E) P values were assessed using two-tailed unpaired t test (∗ P < .05; ∗∗ P < .005) between the Th3 and T-E(M) groups only. N = 3 for each group.