Fig 1: Genetic deletion of Eif4e reduces eIF4E protein expression in p190 cells(A) Established p190 WT and eIF4E+/- leukemia cells have similar immunophenotype characterized by B220 and CD43 expression. Representative flow cytometry plots of p190 cells.(B) Protein analyses of eIF4E, 4E-BP-1, 4E-BP-2 and actin in WT and Het p190 cells. Relative protein quantification performed by densitometric analysis using ImageJ64 software. Data are expressed as mean ± SEM. Fold change was calculated using actin as loading control and normalized to WT sample for each independent experiment. Significance was calculated using unpaired t test (∗p < 0.05; ∗∗∗∗p < 0.001, n = 7).(C) p190s were generated from WT or Het mice and we used a bicistronic dual Renilla-Firefly luciferase reporter construct to measure cap-dependent translation (Renilla luciferase) relative to cap-independent, Coxsackie virus IRES mediated translation (firefly luciferase) as an internal control. MLN0128 (100 nM) treated cells were used as a control. Data are represented as mean ± SEM. Fold change was calculated using WT vehicle condition. Significance was calculated using a paired one tailed student's t test. (∗p < 0.05; ∗∗p < 0.01; n = 3 or 4 per group).(D) Western blots of eIF4E, 4E-BP-1, 4E-BP-2, and total ERK (tERK) in eIF4E+/+, eIF4E fl/+ and eIF4E fl/fl p190 after 72hr of 4OHT (1 μM) treatment.(E) Relative protein quantification performed by densitometric analysis using ImageJ64 software. Fold change calculated using tERK as loading control. Data are expressed as mean ± SEM. Significance was calculated using paired t test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.001, n = 4–6 per group).
Fig 2: CXCR4 expression marks rare clonal HE with HSC potential in the early murine embryo (A) Methodology for index sorting of single P-Sp-derived V+E+61+ cells to co-culture on AGM-ECs and subsequent analysis to assess HSC potential. HSC colony-forming cells (HSC-CFCs) indicate clonal V+E+61+ cells that generate colonies containing HSCs detectable by immunophenotype (VE-cadherin-/low CD45+Gr1-F4/80-Sca1hiEPCRhi, indicated in “HSC gate”), whereas HPC-CFCs lack immunophenotypically detectable HSCs (i.e., no cells detected in the HSC gate).(B) Number of HSC-CFCs and HPC-CFCs detected across seven independent experiments from E9 to E10 (19–30 somite pairs [sp]). Error bars show mean ± SEM. p values indicate unpaired Student’s t test.(C) Expression of CXCR4, CD44, and DLL4 in the VE-cadherin+ population and the V+E+61+ subset of E9.5 (29 sp) P-Sp. Blue histograms indicate isotype controls.(D) Correlation of clonal HSC-CFC and HPC-CFC potential with expression of CXCR4 on individual index-sorted V+E+61+ cells at E9.5 (26–29 sp).(E) Donor-derived peripheral blood (PB) engraftment of clonal progeny of the single HSC-CFCs in (D), including B lymphoid (CD19), T lymphoid (CD3), and myeloid contribution.(F) Correlation of clonal HSC-CFCs and HPC-CFCs with surface expression of CXCR4 and CD41, CD43, and CD45 on individual index-sorted V+E+61+ cells at E9.5 (29–30 sp).(G) Distribution of CXCR4+ and CXCR4- HSC-CFCs and HPC-CFCs detected across six independent experiments from E9 to E10 (19–30 sp). Error bars show mean ± SEM. p values indicate unpaired Student’s t test.(H) Donor-derived PB engraftment of the hematopoietic progeny of bulk-sorted CXCR4+ and CXCR4- cells within the V+E+ population at E9.5 (22–29 sp) following AGM-EC co-culture and transplantation. Error bars show mean ± SEM; n indicates number of recipient mice. All recipients of CXCR4+ cells had multilineage engraftment.See also Figure S1 and Table S1.
Fig 3: Clonal HE harboring multilineage hematopoietic activity emerge independently from HSC-competent HE in the early embryo(A) Methodology for analysis of in vitro hematopoietic lineage potential of clonal progeny of HPC-CFCs. Representative images of erythroid colony-forming unit (CFU-E) (original magnification, ×20), macrophage CFU (CFU-M) (original magnification, ×20), and granulocyte-monocyte CFU (CFU-GM) (original magnification, ×4) shown.(B and C) Correlation of clonal hematopoietic lineage potential with CXCR4, CD41, CD43, CD44, and CD45 expression of individual HPC-CFCs from E9.5 P-Sp (26–28 sp). Multipotent (MPP-CFCs), lymphomyeloid (LMP-CFCs), erythromyeloid (EMP-CFCs), and myeloid colony-forming cells (myeloid-CFCs). No colony/unknown (gray) indicates absence of detectable hematopoietic output following AGM-EC or secondary CFU/OP9 assays.(D) Total progenitor CFCs within the V+E+61+ population per P-Sp detected across five independent experiments from E9 to E10 (19–30 sp). Error bars show mean ± SEM. p values indicate unpaired Student’s t test.See also Figure S2.
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