International Journal of Stem Cells : eISSN 2005-5447

Fig. 4.

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Fig. 4. Transient expression of Lef1 is required for proper differentiation. (A) A6P10 ES cells were infected with retroviruses expressing control or Lef1 shRNA, followed by selection using puromycin. After 2 days without LIF, the selected ES cells were harvested and analyzed by western blotting with anti-Lef1 antibody. β-actin was used as a loading control. (B) 46C ES cells expressing control shRNA or Lef1 shRNA were plated for 7 days in N2B27 medium. GFP fluorescence indicating a neural precursor was not induced in shLef1-46C cells (left panel). The amount of Nanog or Sox1 transcript was measured by RT-PCR analysis (right panel). (C) 46C ES cells were transfected with control vector, Lef1-FL, or Lef1-DN and then selected using Zeocin. 46C ES cells expressing each vector were plated for 7 days in N2B27 medium and GFP fluorescence was captured. (D) RNA samples were isolated from A6P10 ES cells expressing shGFP or shLef1 on the days indicated post-EB formation. Cardiomyocyte (α-MHC) and endoderm (GATA4) markers and loading control (18s rRNA) were used for RT-PCR analysis.
International Journal of Stem Cells 2020;13:192-201
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