GRP94-deficient LSK cells displayed increased proliferation and loss of quiescence.

<p>A) Representative flow cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. To examine early effects of GRP94 depletion on HSC proliferation, BM was extracted from WT and cKO mice 3 days after 4 shots of pI.pC injection every other day. B) Summary of cell cycle distribution of LSK cells from WT and cKO mice (n = 7). C) Summary of flow cytometric analysis of apoptotic LSK cells using Annexin V and 7AAD (n = 5 for WT, n = 8 for cKO) (p = 0.324). All data are presented as mean ± s.e., **p<0.01, ***p<0.001.</p

<i>Grp94</i> KO mice displayed altered myeloid and lymphoid differentiation.

<p>A). Complete blood count of peripheral blood from WT (n = 31) and cKO (n = 37) mice. B) Representative Wright-Giemsa staining of blood smear with tail peripheral blood from WT and cKO mice. Scale bar represents 500 µm. C) Total thymus cell number (left) and total left and right axillary lymph nodes cell number (right) from WT and cKO mice (n = 7 for each group). D) Representative flow cytometric analysis of splenocytes from WT and cKO mice using lineage markers Gr-1 and CD3 (left), F4/80 and B220 (right). E) Quantitation of (D) from WT (n = 4) and cKO (n = 7) mice. F) Representative flow cytometric analysis with BM cells using lineage markers Gr-1 and B220 (left) and CD4 and CD8a (right). G) Quantitation of (F). Gr-1 and B220 (n = 7 for WT and n = 9 for cKO mice); CD4 and CD8a (n = 7 for each genotype). All data are presented as mean ± s.e., ***p<0.001.</p

Conditional knockout of <i>Grp94</i> in the bone marrow.

<p>A) Schematic drawings of the <i>Grp94</i> wild-type (WT) allele, the floxed allele and the knockout (KO) allele. The exons are boxed and numbered. The loxP sites (closed triangle) and the FRT site (open triangle) and expected PCR products for genotyping is indicated. B) Representative BM PCR genotyping results of mice with indicated genotypes after pI.pC injection. C) <i>Grp94</i> mRNA expression measured by quantitative real-time PCR from WT (n = 16) and cKO (n = 18) mouse BM after pI.pC injection. The level of <i>Grp94</i> mRNA was normalized against the level of internal control <i>18S RNA</i>. The data are presented as mean ± s.e., ***p<0.001.</p

Increased mobilization of <i>Grp94</i> KO HSCs.

<p>A) Representative flow cytometric analysis of splenocytes from WT (n = 9) and cKO (n = 9) mice using Lin, c-Kit and Sca-1 (left) and quantitation (right). In these studies, splenocytes were extracted from WT and cKO mice 5 days after 7 injections of pI.pC (to examine HSC mobilization before the spleen was enlarged). B) Representative flow cytometric analysis of blood MNCs from WT (n = 11) and cKO (n = 10) mice using Lin, c-Kit and Sca-1 (left) and quantitation (right). All data are presented as mean ± s.e., ***p<0.001.</p

<i>Grp94</i> KO HSCs displayed impaired interaction with the niche.

<p>A) Scheme of the <i>in vivo</i> competitive lodgment assay. B) Representative flow cytometric analysis of CFSE-labeled WT LK cells and SNARF-labeled cKO LK cells with host BM and spleen cells. C) Summary of LK cells homed to the BM and spleen (n = 4 for BM, n = 2 for spleen), with the level of WT cells in BM and spleen set as 1. D) Bone section of a recipient femur. WT LK cells were labeled with CFSE (green); cKO LK cells were labeled with SNARF (red); and nuclei were stained with DAPI (blue). Solid arrows indicate cells lodged in the endosteal region (within two cell diameters from the endosteal surface), while open arrows indicate cells located in the central marrow. Scale bar represents 1 mm. E) Summary of the percentage of labeled LK cells found at the endosteal region among those homed to BM from 3 independent experiments. All data are presented as mean ± s.e., *p<0.05.</p

Inability of <i>Grp94</i> KO HSCs to express surface integrin α4 and bind to fibronectin.

<p>A) Representative flow cytometric analysis of CD49d and CD49e with BM LSKFlk2⁻ and LSKFlk2⁺ cells from WT and cKO mice. Grey-filled histogram represents isotype control staining; dashed green line represents WT cells; solid red line indicates cKO cells. B) The percentage of WT and cKO LSK cells bound to fibronectin <i>in vitro</i>. The number of cells binding to BSA was subtracted from that binding to fibronectin, the results then were normalized against the number of WT cells bound to BSA. The experiments were performed twice in duplicate; each replicate contained pooled BM from 2 to 4 WT or cKO mice. The data are presented as mean ± s.e..</p

GRP94 deficiency led to increased granulocyte–monocyte progenitors in the bone marrow.

<p>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 ± s.e., *p<0.05, **p<0.01.</p

<i>Grp94</i> KO HSCs failed to reconstitute the hematopoietic system in the presence of WT competitors.

<p>A) Scheme of the <i>in vivo</i> competitive repopulation assay. B) Representative flow cytometric analysis with tail blood from recipient mice 4 weeks after BM transplantation. CD45.1⁺ cells represent blood cells from WT competitor, while CD45.2⁺ cells represent progenies from WT or cKO LSK cells. Tail blood from recipient mice 8, 12, and 24 weeks after BM transplantation yielded the same results (data not shown). C) Summary of the percentage contribution of WT or cKO LSK cells to the peripheral blood MNCs (n = 5). The data are presented as mean ± s.e., ***p<0.001.</p

Increase extramedullary hematopoiesis in the spleen of <i>Grp94</i> KO mice.

<p>A) Representative spleen size and morphology of WT and cKO mice. B) Average spleen weights of WT (n = 19) and cKO (n = 25) mice. The data are presented as mean ± s.e., ***p<0.001. C) Representative H&E staining of paraffin sections of spleen from WT and cKO mice. Hematopoietic cells in the red pulp are indicated by the area between the two arrows. Scale bar represents 2 mm.</p

GRP94 deficiency in the bone marrow expanded the primitive cell pool.

<p>A) Representative flow cytometric analysis with BM cells using Lin, c-Kit, Sca-1, Flk2 and CD34. B) Quantitation of flow cytometric analysis of primitive cell proportions. Left panel shows the percentage of LSKFlk2⁻CD34⁻ in BM and right panel shows LSKFlk2⁻CD34⁺, LSKFlk2⁺ (n = 5 for WT, n = 8 for cKO) and total LSK (n = 16 for WT, n = 22 for cKO) cells in BM using Lin, c-Kit, Sca-1. C) Total BM cell number from WT (n = 19) and cKO (n = 20) mice (p = 0.373). All data are presented as mean ± s.e., **p<0.01, ***p<0.001.</p