Increased mTOR signaling and decreased autophagy in early erythroid progenitors from phlebotomized mtDNA-mutator mice. (A-D) Early erythroid progenitors were sorted from the bone marrow of phlebotomized animals. Cytospins were prepared from the sorted cells and stained with antibodies against phosphorylated 4EBP and LC3. Representative confocal images of phosphorylated 4EBP1 and LC3 staining are shown in panels A and B, respectively. Scale bar represents 2 μM. (C) Mean fluorescence intensity (MFI) of phosphorylated 4EBP1 staining within 4,6-diamidino-2-phenylindole (DAPI)–positive nuclei was calculated for each cell. Graph shows mean MFI ± SEM. n > 15 images per genotype from 3 independent experiments. (D) The number of punctate LC3⁺ structures per cell was counted visually by an observer blinded to the experimental conditions. Graph shows the number of LC3 puncta per cell (circles) and mean value (black line) from a representative experiment. n > 30 cells per genotype. (E) MG staining was analyzed by FACS in the indicated populations of erythroid cells. MFI was normalized to that of Ter119⁺ population “A” in wild-type mice for each experiment. Graphs show mean ± SEM. n = 4 mice per genotype. (F) Schematic diagram highlighting the increase in mTOR activity and decrease in autophagy in early erythroid progenitors from phlebotomized mtDNA-mutator mice. We speculate that the increase in mitochondrial mass in Ter119⁺ population “A” results from the defect in autophagy in early erythroid progenitors.