Disruption of autophagy in erythroid progenitors of mtDNA-mutator mice accelerates the onset and progression of anemia. (A) Survival curve showing percent survival at different time points. W: Atg7^f/fEpor^wt/wtPolgA^wt/wt; A: Atg7^f/fEpor^{wt /GFPCre}PolgA^wt/wt; P: Atg7^f/fEpor^wt/wtPolgA^mt/mt; and D: Atg7^f/fEpor^wt/GFPCrePolgA^mt/mt. (B) Femurs isolated from 11-month-old mice at necropsy. Note the pallor in the double-mutant animals (“D”, arrow). (C-D) Graphs (mean ± SEM) showing hemoglobin (HGB) and mean corpuscular volume (MCV) values in mice at different ages. Summary of statistical analyses for panels C-E are shown in supplemental Table 6. (E-F) PB RBCs were gated based on characteristic forward and side scatter properties and analyzed by FACS for surface CD71 expression and TMRM staining. (E) Graphs showing the percentage of PB RBCs within the R1 and R2 gates from mice at different ages. (F) Representative FACS plots showing the distribution of cells in the R1 and R2 gates from 11-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. (G-J) Ter119⁺ erythroid cells were positively selected from spleens or bone marrows of phlebotomized mice by using magnetic beads. (G) CIV and CS activities were measured and their ratios calculated. n = 5 (W), 4 (A), 4 (P), or 3 (D) mice. (H) Values obtained from ATP analysis were normalized to that of wild-type or PolgA^mt/mt mice for each set of animals. n = 6 (W), 6 (A), 4 (P), or 4 (D) mice. (I) The oxygen consumption rate (OCR) was analyzed by using the Seahorse XF-24 Extracellular Flux Analyzer. Values were normalized to that of wild-type or PolgA^mt/mt mice for each set of animals. n = 3 (W), 2 (A), 6 (P), or 5 (D) mice per genotype. (J) Quantitative PCR analysis was used to determine the ratio of ND2 (mtDNA) to 18S (mtDNA) (an estimate of mtDNA copy numbers per genome). This ratio was normalized to that of wild-type or PolgA^mt/mt mice for each set of animals. n = 4 (W), 4 (A), 6 (P), or 6 (D) mice.