Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation

Figure 3

Nanog is expressed in differentiated cells. (a) Western blot analysis shows endoderm markers and Nanog reexpression after NO treatment. Blots are representative of three independent experiments. C: control cells cultured with LIF for 4 days; T: cells cultured with LIF for 3 days and then exposed to 1 mM DETA/NO for 19 h. Days 5 and 6 refer to DETA/NO treated cells cultured in the absence of LIF for additional periods of 1-2 days. (b) Endoderm markers are expressed in both Oct4 positive and negative cell populations after in vitro differentiation. Real-time PCR analysis for expression of pluripotent (Oct4 and Nanog) and endoderm (Gata4, FoxA2, Cxcr4, Pdx1, Sox17) markers in control (undifferentiated cells grown for 4 days in the presence of LIF), treated and DE differentiated GFP positive and GFP negative cells. D3-Oct4 cells were grown, differentiated, and sorted for GFP expression according to the protocol described in Section 2. Control cells indicate cells grown for 4 days in the presence of LIF; NO-treated cells indicate cells maintained for 3 days in the absence of LIF and treated for 19 h with 1 mM DETA/NO. DE-differentiated cells indicate cells 2 days after treatment in the absence of LIF. Significant difference from GFP⁺; significantly different from control condition. (c) Quantitative real-time PCR for mesodermal lineage markers expressed before and after DETA/NO treatment.

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