Figure 5. Impaired Fbw7-mediated cyclin E control dysregulates mitochondrial mass during terminal erythroid cell maturation in vivo.

(A) Total mitochondrial mass was compared in Ter119+, bone marrow erythroid cells at the indicated maturation stages, based on CD71 vs. FSC. Each histogram is comprised of data obtained from two wild-type and knock-in mice. (B) Transmission electron micrograph images of peripheral blood erythrocytes (RBCs) are shown from mice of the indicated genotypes, with mitochondria retained in cyclin E^{T74A T393A} cells indicated by bars. Peripheral RBCs with retained mitochondria were enumerated by counting approximately 1000 cells from three mice of each genotype. Mean percentage of cells with retained mitochondria and standard deviations are shown, calculated from biological replicates. (C) Bnip3L and Hbb-b1 expression in Ter119+ cells isolated at the indicated stages of erythroid maturation based on CD44/FSC gating is shown for cyclin E^{T74A T393A} mice, compared to age- and sex-matched wild-type controls. (D) Survival of CFSE-labeled erythrocytes obtained from mice of the indicated genotypes was compared by obtaining blood samples from wild-type recipients at the indicated times, and fractions of fluorescent RBCs determined by flow cytometry. The percentage of CFSE-positive RBCs at 12 hours after injection of labeled cells was between 7%–9% of total. Data for subsequent time points are expressed as ratios to 12-hour values and represent averages from four recipients of cells from each genotype. Calculated half-lives are: 19.1 days (wild-type) and 16.5 days (cyclin E^{T74A T393A}), p=0.008. Inset- peripheral blood reticulocytes were enumerated in five wild-type and cyclin E^{T74A T393A} mice, p=0.02.