Figure 4. Oxidative metabolism and mitochondrial ROS are increased in cyclin E^{T74A T393A} erythroid cells.

(A) Oxidative consumption rates coupled to mitochondrial ATP production were assayed in primary Ter119+ cells from three sets of mice of the indicated genotypes using a Seahorse Extracellular Flux Analyzer. (wt= wild-type, T74A T393A= cyclin E^{T74A T393A} cells; ** - 4×10⁵, * - 3×10⁵ cells analyzed in independent assays). Error bars indicate standard error with four replicates per sample. (B) Expression of the indicated genes in purified Ter119+ CD71-high erythroid cells was measured by RT-PCR. Each grey bar represents averaged value of triplicate assays from a single knock-in mouse, expressed relative to expression in cells from age- and sex-matched wild-type controls. Inset shows total and phospho-Rb expression from similarly isolated cells primary erythroid cells. (C) Left column - bone marrow Ter119+ cells were subdivided into distinct morphologic subpopulations (II–V) based on CD71 expression vs. forward scatter (FSC)¹⁴, with relative abundances indicated. Right columns - intracellular reactive oxygen species were measured in the indicated erythroid cell subsets using cell-permeable 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA), enabling detection by flow cytometry. Comparisons between mean fluorescence signals for bone marrow erythroid cells of four mice from each of the indicated genotype groups were made and p-values calculated using paired t-tests. (D) Left – Representative comparison is shown of mitochondrial superoxide levels measured in bone marrow erythroid cells from three pairs of mice at the indicated stage of maturation based on CD44/FSC, using Mitosox Red superoxide indicator. Right –100x micrographs of May-Grunwald/Giemsa-stained orthochromatic erythroblasts isolated using the gating employed for the Mitosox assays. (E) Top – ethidium bromide staining of the indicated mitochondrial DNA (mtDNA) amplification products, resolved on agarose gels, is shown. The mtDNA templates were obtained from Ter119+ bone marrow cells isolated from two mice of each of the indicated genotypes. Bottom – quantification of Ter119+ cell mtDNA amplification for three pairs of mice, performed using PicoGreen reagent, expressed relative to wild-type mtDNA amplification. Derivation of the calculation of for the increase in mtDNA lesions per 10kb is previously described,²⁶ using normalization to 117 base-pair (bp) mtDNA amplification product for mitochondrial copy number.