Figure 2: Delayed erythroid maturation and mitochondrial clearance in vitro.

(a) Colony forming assay for bone-marrow hematopoietic stem cells (Mutator: n=8, WT: n=8, each sample analyzed in triplicate). Mutators showed decreased amount of BFU-E (burst-forming unit, erythroid) colonies (two-tailed P<0.0001, Student’s t-test, data are presented as mean±s.d.). (b) Time required to form haemoglobin-containing mature BFU-Es was delayed by 5 days in Mutators compared with WT (P<0.0001, linear regression analysis). (c) Confocal microscopy of cultured reticulocytes from phlebotomized animals with or without NAC treatment: polarized (TMRM in red) and depolarized (MitoTracker Green) mitochondria. Mutator reticulocytes retained mitochondria longer than WT cells; part of the mitochondria were still polarized after 3 days of culture (arrowheads). Scale bars, 10 μm. (d) Quantification of cells with mitochondria (FACS analysis, MTG per total cell count): Mutator reticulocytes retained mitochondria significantly longer than WT, and NAC had no effect on the clearance (WT versus Mutator day 1 P=0.004, day 2 P=0.002 and day 3 P=0.0003, Student’s t-test). (e) Quantification of cells with polarized mitochondria (FACS analysis, TMRM per total cell count): NAC increased depolarization of mitochondria in Mutator reticulocytes (WT versus Mutator P=0.01, Mutator versus Mutator NAC day 2 P=0.03). For (d,e) WT: n=4, Mutator: n=5, WT NAC: n=6 and Mutator NAC: n=6 (30,000 cells per sample were analyzed). Data are shown as mean±s.d.