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Fig. 3. Tbx3 regulates the proliferation and the cell-lineage determination of
hepatoblasts. (A) CD45+, Ter119+, and
c-Kit+ cells were gated and removed from the initial wild-type and
Tbx3-/- mouse liver tissue specimens (E12.5). The
c-Kit- CD45- Ter119- hepatic epithelial cells
were then fractionated based on c-Met expression. For the in vitro colony
assay, the sorting gate was set for the c-Met+ c-Kit-
CD45- Ter119- cell population. The ratios of
c-Met+ cells in c-Kit- CD45-
Ter119- cells and in unfractionated total cells are shown by the
percentage values outside and within the parenthesis, respectively. Wild-type
cells stained with an isotype control antibody were used as a control.
(B,C) Clonal colony formation upon 5-days single-cell culture of
c-Met+ c-Kit- CD45- Ter119- cells
isolated from wild-type (B) or Tbx3-/- (C) livers.
(D) Numbers of large, medium and small colonies per 150 wells, as
formed by wild-type or Tbx3-/- liver-derived
c-Met+ c-Kit- CD45- Ter119- cells
after 5 days of single-cell culture. The average of three independent
experiments (mean ±s.d.). (E-G) Co-immunofluorescence staining
of Alb and CK7 was conducted for clonal colonies formed by c-Met+
c-Kit- CD45- Ter119- cells isolated from
wild-type (E,F) and Tbx3-/- (G) livers after 18 days of
culture. Insets denote individual cells labeled with DAPI. (H)
Following co-immunofluorescence staining of Alb and CK7, the percentage of
Alb+, CK7+, Alb/CK7+, and Alb/CK7-
cells in each colony was determined. The average of 24 colonies (mean
±s.d.). Scale bars: 500 µm in B,C; 100 µm in E-G.
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