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.