Figure 3: Nkx2-5 overexpression in ES/EB system results in increased levels of Tdgf1. (A) Integration of Nkx2-5-expressing vector, pLoxNkx2-5, into the X chromosome of the Ainv15 ES cells to place the cDNA of Nkx2-5 under the control of tetracycline responsive element (TRE). Upon addition of doxycycline (+ Dox), reverse tetracycline transactivator (rtTA) binds to the TRE and induces Nkx2-5 expression. (B) Increased expression of Nkx2-5 RNA and protein in EB cells following the induction with Dox for 48hrs is confirmed; *, p<0.05, n=3. (C) Analyses of fold change in the expression of the Tdgf1 transcript using qRT-PCR following the induction with doxycycline (Dox) for 48hrs (day 2-4) of EB formation; *, p<0.05, n=3. (E) Western blot analysis confirms increased expression of Tdgf1 in EBs overexpressing Nkx2-5 following the induction with Dox for 2 days (day 2-4) vs. control EBs.