Figure 1.

mtDNA-mutator mice show a defect in the clearance of mitochondria and ribosomes during terminal stages of erythroid maturation. (A-D) Peripheral blood (PB) RBCs were gated based on characteristic forward and side scatter properties and analyzed by fluorescence-activated cell sorting (FACS) for surface CD71 expression and TMRM staining. (A) Representative FACS plots showing the distribution of cells in the R1 and R2 gates from 12-month-old mice. The R2 gate consists of reticulocytes that are positive for CD71 and contain mitochondria. The R1 gate contains an abnormal population of cells that have lost surface CD71 expression but retain mitochondria (red arrow). (B) Percentage of PB RBCs (mean ± standard error of the mean [SEM]) within the R1 and R2 gates from mice at different ages. n ≥ 4 mice per genotype per time point. *P < .001 (1-way analysis of variance followed by Holm-Sidak post hoc analysis). (C) Percentage of PB RBCs (mean ± SEM) within the R1 and R2 gates from mice before (day “0”) and on the indicated days after completion of the 5-day phlebotomy protocol (“postphlebotomy”) used to induce stress erythropoiesis. n = 3 mice per genotype. (D) Representative FACS plots showing the distribution of cells in the R1 and R2 gates from mice 7 days postphlebotomy. The R1 gate contains an abnormal population of cells that have lost surface CD71 expression but retain mitochondria (red arrow). (E) Representative images from fluorescence and differential interference contrast microscopy of PB RBCs collected 5 days postphlebotomy and stained with MG and TMRM. Scale bar represents 10 μM. (F) Representative FACS plots showing the profile of CD71 expression and MitoTracker Red staining in PB RBCs collected 7 days postphlebotomy. Cells were gated based on CD71 expression and analyzed for thiazole orange staining. The retention of RNA and ribosomes in CD71^low/med cells from mtDNA-mutator mice is highlighted by the red arrows.