Page 98 - Haematologica April 2020

S. Altamura et al.
sis of blood parameters using a Sysmex XT-2000iV apparatus as described.41
Vitamin E depletion and repletion
Mice were deprived from vitamin E by feeding a vitamin E-depleted diet (SSNIFF, Soest, Germany) containing 7ppm α-tocopherol as compared to 55ppm in the normal diet. In initial experiments the conditions for ruling out side effects of Cre acti- vation33 with and without vitamin E-depletion using Gpx4wt/wt;CreERT2 mice as controls were defined (Online Supplementary Figure S1). This allowed to apply a simplified proto-
col with C57BL/6J mice as controls and tamoxifen-treated, lethally irradiated wt mice reconstituted with BM cells of Gpx4fl/fl;CreERT2 mice receiving a normal or a vitamin E-depleted diet as experi- mental groups (Figure 2-5 and Online Supplementary Figure S5). The vitamin E-repleted diet (5x vitamin E) contained 275ppm α-tocopherol (SSNIFF, Soest, Germany).
Staining of erythroid precursor cells from BM and spleen
Spleens were smashed and washed and the four leg bones flushed with 5 mL PBS containing 1mM EDTA and 2% fetal calf
ABC
DEF
GHI
JKL
Figure 2. Relative increase in immature erythroid precursor cells in the bone marrow and spleen of mice with Gpx4-deficiency in the hematopoetic system. Representative fluorescence-activated cell sorting (FACS) staining of (A-F) BM and (G-L) spleen cells with CD44-PE-Cy7 and Ter119-PE (A-C) as well as with CD71- PE-Cy7 and Ter119-PE antibodies (D-F, J-L). The gate set by forward sideward scatter (FSC) and lineage marker-negative cells (G-I) illustrates the increase in extramedullary erythropoiesis in the spleen of mice with Gpx4-deficient hematopoietic cells kept on a (H) normal or (I) on a vitamin E-depleted diet as compared to (G) Gpx4 wt mice kept on a normal diet. The shift towards immature erythroid precursor cells is strongly increased under combined Gpx4- and vitamin E-deficiency (C, F, L).
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haematologica | 2020; 105(4)