DEV016634 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Tbx3 expression in the developing mouse liver. (A-U) Immunofluorescence staining of Tbx3 (A,E,I,M,P,S) and Hnf4α (B,F,J,N,Q,T) in E9.5 (A-H), E10.5 (I-L), E14.5 (M-O), E16.5 (P-R) and neonatal liver (S-U). Magnified pictures of A-D are shown in E-H, respectively. Arrowheads indicate some cells still expressing Tbx3 weakly in E16.5 liver. DNA is stained with DAPI. (V) Tbx3 expression in E12.5, E13.5, E15.5, E17.5 and neonatal liver was examined by qPCR. All data were normalized to the values of E12.5 liver and fold differences are shown. Bar represents mean ±s.d. (n=3). Scale bars: 100 µm in A-L; 25 µm in M-U.

• Supplemental Figure S2 -

  Fig. S2. Decrease in the number of proliferating Hnf4α<^b>⁺ cells in the Tbx3<^b>⁻<^/b>^/<^b>⁻ embryonic liver. (A-H) Immunofluorescence staining of Hnf4α (A,E) and PCNA (B,F) for wild-type (A-D) and Tbx3^−/− (E-H) liver (E12.5). DNA is stained with DAPI. Scale bar: 100 µm.

• Supplemental Figure S3 -

  Fig. S3. Apoptosis is not induced in the Tbx3<^b>⁻<^/b>^/<^b>⁻ embryonic liver. Immunofluorescence staining of cleaved caspase 3 for wild-type (A,C) and Tbx3^−/− (B,D) liver (E12.5). We examined 7-10 sections of each wild-type (n=3) and Tbx3^−/− (n=3) embryo. Representative data are shown. DNA is stained with DAPI. Scale bar: 100 µm.

• Supplemental Figure S4 -

  Fig. S4. Suppressing Tbx3 expression in progeny of a single hepatoblast inhibits proliferation and promotes cholangiocyte differentiation. (A) Experimental procedures to elucidate functions of Tbx3 in hepatoblasts. Upon single-cell culture of c-Met⁺ c-Kit⁻ CD45⁻ Ter119⁻ cells isolated from E13.5 wild-type livers, cells propagating in culture were replated onto new culture dishes and then transfected with constructs harboring Tbx3-shRNA and a puromycin-resistance gene. As reported, such hepatoblast progenies maintained an immature hepatic cell population by self-renewing cell divisions, as well as giving rise to hepatocytes and cholangiocytes as descendants (Suzuki et al., 2002). After selecting stable transfectants with puromycin, their potential for proliferation and differentiation was analyzed. Vehicle-transfected cells were used as a control. (B) Immunofluorescence staining of Tbx3 for progeny of a single hepatoblast isolated from the c-Met⁺ c-Kit⁻ CD45⁻ Ter119⁻ cell population. DNA is stained with DAPI. (C) Western blot analysis of Myc-Tag (Tbx3) in cultured hepatoblasts carrying Myc-Tbx3 constructs with vehicle or Tbx3-shRNA constructs. Tbx3-shRNA efficiently downregulates the level of Myc-Tbx3 protein. (D) p19^ARF expression in cultured hepatoblasts carrying control or p19^ARF-siRNA was examined by qPCR. p19^ARF-siRNA efficiently downregulates the level of p19^ARF expression. All data were normalized to the values of cultured hepatoblasts carrying control siRNA and fold differences are shown. Bar represents mean ±s.d. (n=3). (E) Numbers of cells per well of type-IV-collagen-coated 6-well plates at days 3 and 5 of culture after plating 1×10⁴ cells of each transfectant. Additional transfection of p19^ARF-siRNA into Tbx3-shRNA-expressing hepatoblasts partially rescued a growth defect in the Tbx3-shRNA transfectant. Bar represents mean ±s.d. (n=3). (F,G) Co-immunofluorescence images of Alb and CK7 in colonies formed by each transfectant. DNA is stained with DAPI. (H) Gene expression analysis by qPCR for each transfectant. All data were normalized to the values of vehicle-transfected cells and fold differences are shown. Bar represents mean ±s.d. (n=3). Scale bars: 50 µm in B,F,G.