Zhou et al. 10.1073/pnas.0702409104.Supporting Information

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SI Figure 4
SI Figure 5
SI Figure 6


SI Figure 4
Fig. 4. B cell precursors in the bone marrow of wild-type C57BL/6 mice. Single-cell suspensions of bone marrow cells were prepared and stained with antibodies against B220, CD43, and IgM, followed by FACS sorting. B cell precursors were defined as pro-B (B220+CD43+IgM-) (A), pre-B (B220+CD43-IgM-) (B), or immature B cells (IMB) (B220+CD43-IgM+) (B). (C) Splenic mature B cells (B220+IgM+IgD+) were isolated from the spleen of a normal C57BL/6 mouse.


SI Figure 5
Fig. 5. Ectopic expression constructs. Authentic stem-loop and flanking sequences of various lengths on the 5' and 3' side for miR-150 and miR-195 were cloned into the MDH.xdna murine retroviral vector. miRNA ectopic expression constructs and the empty MDH vector were individually transfected into 293T cells. Forty-eight hours after transfection, total RNAs were isolated and loaded onto a 10% denaturing polyacrylamide gel. DNA oligo probes that were complementary to each of the selected miRNAs were labeled and hybridized to the membrane to detect mature miRNAs that can be efficiently processed (20- to 24-nt). miR-150-90nt and miR-150-130nt represent retroviral constructs containing miR-150 pre-miRNA and 90 or 130 nt of flanking sequences on either side of the stem-loop sequences, respectively. miR-195-90nt and miR-195-140nt represent retroviral constructs containing miR-195 premiRNA sequence with 90 or 140 nucleotides flanking sequences on either sides of the stem-loop sequences, respectively. Constructs with high processing efficiency were selected for bone marrow transplantation.


SI Figure 6
Fig. 6. Short-term repopulation analysis of peripheral blood samples. Peripheral blood samples were collected at 4 weeks after transplantation from mice that were transplanted with hematopoietic stem/progenitor cells infected with miR-150, miR-195, or control MDH retroviral supernatant, also expressing GFP. Erythrocytes were depleted using ammonium chloride solution. Cells were then stained with antibodies against the donor cell surface marker (CD45.2) and against cell lineage-specific surface markers (CD4, CD8, CD19, CD11b, and Gr-1), followed by FACS analysis. The FACS plot of the donor derived (CD45.2+) cells in the blood of one mouse from each group is displayed.