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Fig. 4. (A) TRE-Nanog ES cells were plated on gelatin-coated plate and cultivated for 3 days in the presence or absence of LIF and doxycycline (DOX, 1 µg ml–1). In the absence of DOX, Nanog transgene was overexpressed in the cells (tet-off system). Nanog-overexpressed ES cells maintained undifferentiated morphology regardless of the presence of LIF. Bars, 500 µm. (B) Morphology of TRE-Nanog ES-cell aggregates. TRE-Nanog ES cells were subjected to aggregation culture in the presence or absence of LIF and DOX. The phase-contrast images were captured under a light microscope after 4 days of aggregation. Bars, 200 µm. (C) Overexpression of Nanog inhibited visceral endoderm gene expression in the aggregates. Isolated total RNA from each condition in B was subjected to RT-PCR analysis. PCR products were separated on a 2% agarose gel and visualized by ethidium-bromide staining.
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