International Journal of Stem Cells : eISSN 2005-5447

Fig. 3.

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Fig. 3. Ectopic expression of Tcf1 maintains self-renewal and delays differentiation of ES cells. (A) mRNA expressions for Oct4, Sox2 and Nanog at ES cells stage and Day 4 of differentiation upon TCF1 overexpression were determined by using qPCR. RNA was isolated from cells expressing empty vector (EV) or Tcf1-FL (TCF1) on specified stage. GAPDH was used for normalization. (B) The amount of Nanog transcript was measured by RT-PCR analysis. RNA was isolated from ES cells expressing control vector or Tcf1-FL on the days specified. 18s rRNA was used as a loading control. (C) ChIP assay was performed to examine the binding of Tcf1, Lef1, and Tcf3 on Nanog promoter. Samples for ChIP were isolated from ES cells grown with LIF and from ES cells expressing control vector or Tcf1-FL cultured for 2 days without LIF. For immunoprecipitiation, endogenous antibody against Tcf1, Lef1, or Tcf3 was used. (D) To differentiate into neural precursors, 46C ES cells expressing control vector, Tcf1-FL, or Tcf1-DN were plated for 6 days in N2B27 medium. GFP fluorescence showed that 46C ES cells expressing control vector or Tcf1-DN, but not Tcf1-FL effectively differentiated into neural precursor (upper panel). The amount of Nanog or Sox1 transcript on the days indicated was measured via RT-PCR analysis (lower panel). β-actin was used as a loading control. (E) RNA samples were isolated from A6P10 ES cells expressing control vector or Tcf1-FL on the days indicated after EB formation. Self-renewal (Nanog), mesoderm (Brachyury), and cardiomyocyte (Islet1, αMHC) markers were used for RT-PCR analysis.
International Journal of Stem Cells 2020;13:192-201 https://doi.org/10.15283/ijsc20044
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