GRP94 Regulates Stem Cell-Niche Interactions Figure 3. GRP94 deficiency led to increased granulocyte–monocyte progenitors in the bone marrow. A) Quantitation of flow cytometric analysis of myeloid and lymphoid progenitors including common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), megakaryocyte-erythroid progenitor (MEP) and common lymphoid progenitor (CLP) from WT (n$7) and cKO (n$10) mice. B) Quantitation of colonies formed from unfractioned BM from WT (n = 4) and cKO (n = 7) mice in methylcellulose medium. All data are presented as mean 6 s.e., *p,0.05, **p,0.01. doi:10.1371/journal.pone.0020364.g003 (Figure 4A). While approximately 75% of the LSK cells in both genotypes were in G1 phase, the proportion of cells in G0 decreased from 15% in the control to 7.7% in the cKO mice (p,0.001), whereas the amount of S+G2+M cells increased from 10% in the control to 17% for cKO mice (p,0.01) (Figure 4B). Furthermore, the expansion of the Grp94 KO primitive hemato- poietic cell pool was not a consequence of altered cell survival as analysis of the level of apoptotic cells using Annexin V did not reveal significant differences between WT and cKO LSK cells (p = 0.324) (Figure 4C). These findings suggest a loss of quiescence and increase in proliferation account at least in part for the primitive hematopoietic cell expansion observed in cKO mice, similar to what has been shown using other models of HSC function [45]. Grp94 KO HSCs display increased mobilization and impaired HSC-niche interaction We next examined whether the alteration in the cell cycle status of the primitive hematopoietic cells was accompanied by any changes in their localization [46]. Histological analysis showed that the spleen size was significantly enlarged in cKO mice PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e20364