Fas Stabilizes and Accelerates Erythropoiesis Figure 8. Delayed response to hypoxia-induced stress in mice deficient in the Fas pathway. (A–D) gld-Rag12/2 or control mice were transferred to a hypoxia chamber with ambient 11% oxygen for 1 or 3 days. Data is mean 6sem for 4 to 9 age and gender-matched mice per time point per genotype. Data at t = 0 is the basal state data shown in Figures 3A and 5A, pooled for males and females. (A) Hematocrit measurements, performed via CritSpin microcentrifugation. (B) Reticulocyte measurements, performed flow cytometrically, as in Figure 3C. (C) Plasma Epo, measured by ELISA. (D) Spleen erythroid subsets ProE, EryA, EryB and EryC, expressed as absolute number of cells per gram body weight in gld-Rag12/2 and matched Rag12/2 controls. For all panels: *p,0.05, **p,0.005, ***p,0.0005 (t test, unequal variance). doi:10.1371/journal.pone.0021192.g008 The increase in spleen ProE, EryA and CFU-e is due to the absence of a spleen-specific negative regulator, rather than erythropoietic stress, since it is not associated with elevated serum Epo, and since there is no associated increase of bone-marrow progenitors. It is unlikely that the increase in ProE/EryA is a consequence of expansion in an earlier, Fas-regulated progenitor compartment, since there was no significant change in bone- marrow erythroid progenitors, platelets or white cells (Table S1). An earlier report of increased CFU-S in adult lpr and gld mice [50] is complicated by the autoimmune syndrome in these mice, which in the present work we addressed by breeding the lpr and gld mutant mice onto the immune-deficient Rag12/2 background. The expansion in ProE and EryA precursor pools in the gld- Rag12/2 and lpr-Rag12/2 mice represents a substantial non- redundant negative regulatory function of Fas. Nevertheless, it is likely to be an underestimate of the actual number of ProE/EryA that are regulated by Fas in wild-type mice, as suggested by the response to stress (see below). Compensatory mechanisms that PLoS ONE | www.plosone.org 11 July 2011 | Volume 6 | Issue 7 | e21192