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Rheb1 regulates HSC proliferation & differentiation
opsonized with 10% mouse serum for 30 min at 37oC. Neutrophils and bacteria were then incubated together at a 1:5 ratio for 30 and 120 min at 37oC with intermittent shaking, and then 100 g/mL gentamicin was added for an additional 30 min to eliminate extracellular bacteria. The cells were then lysed by adding distilled water, and subsequently diluted aliquots were spread on LB agar plates. The CFU was counted after incubating the plates overnight at 37oC. A bacterial suspension without any cells was used as an input control.
Mouse colony-forming cell assay and 3BDO treatment
3BDO was dissolved in DMSO at 60 mM. WBMCs were treated with 3BDO (60 nM) or DMSO for 30 min.18 Then, GMP/CMPs (2x104 cells) were sorted and cultured in MethoCult® media (Catalog #03231) in the presence of SCF (50 ng/mL), IL3 (10 ng/mL), and GM-CSF (25 ng/mL). Colonies were counted after 5-7 days.
Serial colony-forming cell assay
Whole BM cells were treated with 3BDO (60 nM) or DMSO for 30 min, then cells (1x105) were isolated and cultured in MethoCult® media (Catalog #03434). Colonies were counted seven days after plating, and serially replating. Colonies are defined as colony forming unit monocyte (CFU-M), granulocyte- macrophage (CFU-GM), and granulocyte-eythroid-macrophage (CFU-GEM).
Cobble stone area-forming cell assay
Mesenchymal stem cells (MSC) were obtained from mouse BM cells, as previously described.19 MSCs were cultured in long-term culture medium M5300 (Stem Cell Technologies, Vancouver, BC, Canada) for two weeks, and half of the medium was changed every 3-4 days. Cells were trypsinized, irradiated with 15 Gray, and plated at 5000 cells/well in 96-well plates. LKS+ cells (500 cells/well) were inoculated on the irradiated cell layers and incu- bated at 33°C. Colonies (Cobblestone areas) were counted five weeks after plating.
Gene expression profiling and patient data analysis
LKS+ cells were sorted from WBMCs by BD Influx flow cytome- ter. The sorted cells were treated with Trizol, and then sent to the Shanghai Biotechnology Corporation for microarray analysis (Agilent, mouse 4*44K). Analysis of gene expression data from Rheb1Δ/Δ and Rheb1fl/fl LKSs revealed 2515 differentially expressed genes, with 922 up-regulated and 1593 down-regulated genes in Rheb1-deficient LKSs. The differentially expressed genes were fil- tered as P<0.05 and fold change greater than 3. The gene-set enrichment data were analyzed using GSEA and GO (FDR <0.25, P<0.05, -Lg p = -Log10 p). The microarray data were deposited with the GEO under the accession number GSE79538. For the GSEA analysis of patient data, we examined AML-related data using Molecular Signatures Database (MSigDB) (http://software.broadinstitute.org/gsea/msigdb/search.jsp).
Quantitative real-time PCR (qRT-PCR)
Total RNA from BM samples was extracted using the RNeasy Mini Kit (Qiagen). First-strand cDNA was synthesized with an oligo-(dT) primer according to the manufacturer’s instructions. The mRNA expression was determined by qRT-PCR (Stepone Fast Real-Time PCR system; Applied Biosystems) using FastStart Universal SYBR Green PCR Master mix (Roche). GAPDH was used as endogenous controls for gene expression assays.
Statistical analyses
Experimental results were analyzed using Student t-test. P<0.05 was considered significant for all tests. Kaplan-Meier survival
curves were created using GraphPad Prism 5. All data are present- ed as mean±Standard Error of Mean (SEM); n ≥3.
Results
Rheb1 deletion induced hematopoietic stem cell / hematopoietic progenitor cells expansion in steady state condition
To clarify the role of Rheb1 in hematopoiesis, Vav1- cre;Rheb1fl/fl (Rheb1Δ/Δ) mice were generated and the cellular composition of the peripheral blood (PB) and BM was first analyzed in Rheb1fl/fl and Rheb1Δ/Δ mice. There was no sig- nificant difference in normalized values of white blood cells (WBC), RBCs, hemoglobin (Hgb), hematocrit (Hct) and platelets (PLT) in the PB between Rheb1fl/fl and Rheb1Δ/Δ mice, while absolute numbers of neutrophils were sub- stantially higher in Rheb1Δ/Δ mice than those in Rheb1fl/fl mice (Online Supplementary Figure S1A). The absolute num- bers of BM cells did not alter between Rheb1fl/fl and Rheb1Δ/Δ mice (Online Supplementary Figure S1B). The percentage and absolute numbers of LKS+ and LKS– cells were increased in Rheb1Δ/Δ mice (Online Supplementary Figure S1C and D). In the HSC-enriched LKS+ subsets, Rheb1 loss sig- nificantly increased the absolute numbers of CD150+CD48–LKS+ cells (Figure 1A and Online Supplementary Figure S1E), while in the hematopoietic pro- genitor cells (HPC)-enriched LKS– subsets, Rheb1 loss resulted in an increase in the absolute number of CD16/32–CD34+LKS– cells and CD16/32–CD34+LKS– cells (GMP and CMP) (Figure 1B and C, and Online Supplementary Figure S1F). More Rheb1Δ/Δ LKS+ cells were in G2/S/M phase of cell cycle than that of the control (Figure 1D and Online Supplementary Figure S1G), although the percentage of Annexin V-positive cells was not altered in Rheb1Δ/Δ LKS+ (Online Supplementary Figure S1H). Thus, Rheb1 deletion led to an expansion of murine HSC/HPCs.
Rheb1 deletion caused neutrophil immaturity in steady condition
Interestingly, the absolute numbers and percentages of myeloid cells (CD11b+) were increased in BM of Rheb1Δ/Δ mice (Online Supplementary Figure S2A and B), while no sig- nificant changes were found in B (B220+) and T (CD3+) cells in the BM of Rheb1fl/fl and Rheb1Δ/Δ mice (Online Supplementary Figure S2C). We then analyzed neutrophils by flow cytometry (FACS) with CD11b and Ly-6G anti- bodies that have been used as neutrophil subpopulation markers for the identification of myelocytes/promyelo- cytes, as well as immature and mature neutrophils. The CD11b+Ly-6G+ subpopulation of Rheb1Δ/Δ neutrophils shifted to the left in the PB and BM (Figure 1E). We divid- ed neutrophils into three distinct subpopulations, indicat- ed as the CD11blowLy-6Glow population (P1), the CD11bhigh Ly-6Glow population (P2), and the CD11b+ Ly-6Ghigh popula- tion (P3). We found that the percentages of the P1 and P2 subpopulations were increased, while the percentage of the P3 subpopulation was decreased both in the PB and BM of Rheb1Δ/Δ mice (Figure 1F and G). Morphological analysis of Rheb1fl/fl neutrophils from BM showed that the P1 subpopulation was comprised mainly of myeloblasts with oval-shaped nuclei and a wider, less basophilic cyto- plasm. The segmentation of the nuclei became gradually evident in the P2 subpopulation while the P3 subpopula- tion was mainly composed of cells with doughnut-shaped
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