Tr4 heterozygous mutation results in decreased erythroid cell proliferation without altering apoptosis. Colony-forming assays were performed by plating lineage negative bone marrow cells in erythroid MethoCult (M3334; see “Materials and methods”), imaged, (A) the number of erythroid colony-forming unit (CFU-E) colonies quantified after 3 days and (B) burst-forming unit (BFU-E) colonies after 14 days. (C,D) Bone marrow cells were isolated from WT and Tr4^+/− femurs and stained with α-CD71, α-TER119, α-CD44 (as in Figure 2), and Annexin V antibodies (to assess apoptosis), and the total percentage of cells within each gate were quantified. (E,F) Bone marrow cells were stained with α-CD71, α-TER119, α-CD44, and α-Ki67 antibodies (to assess proliferation), and the total percentage of cells within each gate was quantified. (G) Stage III (CD71⁺TER119⁺) erythroid cells were stained with α-Ki67 and DAPI antibodies. Gating for stages of proliferation are indicated on the left and the percentage of cells within each gate is graphed on the right. *P ≤ .05 as determined by Student t test comparing Tr4 heterozygous mutant with WT bone marrow cells.