Figure 5.

Increased mTOR signaling and decreased autophagy at late stages of erythroid maturation in phlebotomized mtDNA-mutator mice. (A-F) Ter119⁺ erythroid cells were positively selected from spleens of phlebotomized mice by using magnetic beads and sorted by FACS based on CD71 expression and forward scatter. (A-B) Extracts prepared from sorted populations were subjected to immunoblot analyses using antibodies against phosphorylated S6 (pS6), total S6 (S6), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative immunoblots and graphs (B) show higher phosphorylation of ribosomal protein S6 in Ter119⁺ erythroid population “B” (CD71⁺ FSC^low) from mtDNA-mutator mice than from wild-type littermates. n = 2 (wild-type) or 3 (PolgA^mt/mt) mice. (C-D) Sorted populations of erythroid cells were incubated in media containing ³⁵S-labeled amino acids to assess translation. Representative autoradiographs (C) and graphs (D) show higher levels of translation of a 15-kDa protein (consistent with hemoglobin subunit α) in cells from mtDNA-mutator mice. n = 2 (wild-type) or 3 (PolgA^mt/mt) mice. (E-H) Extracts prepared from the sorted populations were subjected to immunoblot analyses using antibodies against LC3 and GAPDH. Representative immunoblots (E,G) and graphs (F,H) show lower LC3 conversion in Ter119⁺ erythroid populations “B” (CD71⁺ FSC^low) and “C” (CD71⁻FSC^low) from mtDNA-mutator mice than in those from wild-type littermates. n = 4 mice per genotype. (I-J) Extracts prepared from Ter119⁺ erythroid population “C” were subjected to immunoblot analyses using antibodies against P62 and GAPDH. Representative immunoblots (I) and graph (J) show increased steady-state levels of P62 in erythroid cells from mtDNA-mutator mice. (K) MG staining was analyzed by FACS in Ter119⁺ erythroid population “C.” MFI was normalized to that of Ter119⁺ population “A” in wild-type mice for each experiment (see Figure 4E). n = 4 mice per genotype. The graph shows the increased mitochondrial content in erythroid cells from mtDNA-mutator mice. Graphs in panels B, D, F, and G show mean ± SEM. P < .05. (L) Schematic diagram highlighting increase in mTOR activity and translation and decrease in autophagy in late erythroid progenitors (Ter119⁺ population “B” or “C”) of aged or phlebotomized mtDNA-mutator mice.