Page 101 - Haematologica April 2020

GPX4 controls reticulocyte maturation
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Figure 5. (A-C) Gpx4-deficiency in hematopoietic cells causes a reticulocyte maturation defect that is to a large extent compensated by vitamin E in vivo. Representative fluorescence-activated cell sorting (FACS) stainings of peripheral blood cells of (A) Gpx4 wt mice kept on a normal diet and of mice with Gpx4-defi- cient hematopoiesis maintained either on (B) a normal or on (C) a vitamin E-depleted diet (C), stained with Mitotracker Deep Red (MTDR), thiazol orange (TO), and CD71-PE-Cy7 and Ter119-PE antibodies. The experiment is described and quantitatively evaluated in the Online Supplementary Figure 5. Ter119-positive cells were gated and plotted as shown in A-C. Immature reticulocytes (CD71high) are shown in green, mature reticulocytes (CD71low) in blue and erythrocytes (CD71neg/Ter119+) in purple. CD71high cells were subdivided into immature and highly immature reticulocytes based on MTDR and TO staining. (C) Under combined Gpx4- and vitamin E-deficiency the fraction of highly immature reticulocytes was strongly increased. D-F) Lipid peroxidation is increased in Gpx4-deficient reticulocytes and erythro- cytes. Peripheral blood cells of the mice shown in A-C mice were stained with anti-CD71-PE-Cy7- and anti-Ter119-APC-Cy7-antibodies and with 2 μM C11- Bodipy(581/591). Ter119-positive cells were gated and the increase in % green fluorescence-positive cells was measured in CD71high cells (immature reticulocytes, D), in CD71low cells (mature reticulocytes, E) and in CD71neg cells (erythrocytes, F) upon excitation at 488 nm. Wild-type, normal diet, (n=2); k.o., normal diet (n=2); k.o., minus vitamin E (n=3). The significance was calculated using an unpaired T-Test. Note the high degree of lipid peroxidation in Gpx4-deficient immature reticu- locytes upon feeding a vitamin E-depleted diet. G-L) Ultrastructural analysis of red blood cells from the mice shown in A-C. Remnants of mitochondria (Mi) are marked by white arrows. Under Gpx4-deficiency (H and K), and more so under combined Gpx4- and vitamin E-deficiency (I and L) large unphagocytosed vesicles containing mitochondria accumulated in reticulocytes (R). Blood pellets were processed for transmission electron microscopy as described in the Online Supplementary Materials and Methods.
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