Figure 3: Mutator erythrocytes show abnormal iron loading and extensive oxidative damage.

(a) Transferrin receptor (red) localization in reticulocytes (Ter119 green) at the second day of culture analyzed by confocal microscopy. WT reticulocytes: most of the TfR was clustered (arrows); Mutator reticulocytes: TfR showed even membrane distribution (dashed circle), even in morphologically mature erythrocytes (arrowhead). NAC treatment rescued TfR distribution in Mutators. WT: n=3, Mutator: n=3, WT NAC: n=3 and Mutator NAC: n=3, samples were analyzed in three separate experiments. Scale bars, 10 μm. (b) Total serum iron level at the age of 10 months (two-tailed P=0.001, Student’s t-test). WT: n=6, Mutator: n=17. (c) Transferrin (Tf) saturation at 10 months of age (two-tailed P=0.006, Student’s t-test). WT: n=6, Mutator: n=11. (d) Free, non-protein-bound iron (NPBI) in erythrocytes (two-tailed P=0.0002, Student’s t-test) and in plasma (two-tailed P<0.0001, Student’s t-test) at the age of 8–10 months (WT: n=7, Mutator: n=7, three to five animals were pooled in each sample). (e) 4-hydroxynonenal (4-HNE) protein adducts, lipid peroxidation-induced protein damage, in the erythrocyte membranes at 8–10 months of age (two-tailed P<0.0001, Student’s t-test). Representative Western blots shown on left and quantification on right (WT: n=7, mut: n=5, three to five animals were pooled in each sample). For b–e, data are shown as mean±s.d.