Abnormal erythroid development in aging Polg^A mice. Polg⁺ and Polg^A mice were humanely killed for analysis at the indicated ages. Single-cell suspensions were prepared from bone marrow (BM) and spleen (Spleen) without hypotonic lysis, then stained for surface expression of CD71 (transferrin receptor) and the erythroid-specific antigen Ter119. In panels A and B, the individual graphs presented are representative of 3 independent experiments for each age group. In each independent experiment, one Polg^A mouse was compared with one or 2 Polg⁺ littermates. The percentages shown with each region represent the mean frequency of that population, as a percentage of all singlet events, across all 3 experiments for the indicated age group. Each region was analyzed for statistically significant differences in frequency between genotypes using the Student t test (*P < .05, **P < .01). (A) All events remaining after exclusion of debris and nonsinglet events (80%-90% of all events; data not shown) were analyzed on the basis of expression of CD71 and Ter119. (B) The regions indicated in panel A were further analyzed on the basis of FSC, a surrogate for cell size relative to expression of CD71. (C) After the exclusion of both lineage⁺ and nonviable cells (see “Flow cytometric analysis” in supplemental Methods) as well as gating as described in panel A, cells from 5 populations were sorted by use of the indicated regions. Sorted cells were then spun on slides, stained with Wright-Giemsa, and visualized. All images were acquired at identical magnification; bar represents 10 microns. (D) After preparation and analysis as described in panel A, spleen and bone marrow from 6-month-old β-thalassemia (bottom left) and WT littermate control (top left) mice were analyzed as in panel B in parallel with samples from 10-month-old Polg⁺ (top right) and Polg^A (bottom right) mice. These results from a single parallel experiment are representative of results obtained during independent analysis of β-thalassemia and Polg D257A samples (3 and 7 samples, respectively). (E) Whole bone-marrow cell suspensions from 10-month-old Polg⁺ (left) and Polg^A (right) mice prepared as in (C) demonstrate the presence of abnormally hemoglobinized orthochromatic normoblasts (arrows) in Polg^A marrow only and normal polychromatic normoblasts (arrowheads) in both Polg⁺ and Polg^A marrow. All images were acquired at identical magnification; bar represents 10 microns.