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Fig. 2 | Stem Cell Research & Therapy

Fig. 2

From: Tcf7l1 directly regulates cardiomyocyte differentiation in embryonic stem cells

Fig. 2

Temporally controlled ablation of Tcf7l1 impaired cardiomyocyte formation without affecting germ layer differentiation. a Genetic elements in Tcf7l1-tetoff ESCs. Two transgenes introduced into Tcf7l1−/− ESCs: tetracycline-controlled transactivator (tTA) driven by CMV promoter and Tcf7l1 driven by tetracycline responsive promoter (TRE-CMV). Presence of tetracycline or doxycycline blocks activity of tTA transactivator, hence silencing Tcf7l1 transgene. b Western blot confirmation of Tcf7l1 transgene silencing by supplemental dox. c Scheme of dox supplementation. d Ablation of Tcf7l1 at days 2 and 4 impaired cardiomyocyte formation, evidenced by reduced positive staining for α-Actinin. e Cardiac mesoderm marker Mesp1, cardiac transcription factors Tbx5 and Nkx2–5, and cardiac structural gene αMHC reduced by Tcf7l1 ablation at days 2 and 4, but not ablation at days 6 and 8. f Nascent mesendoderm genes T, Eomes, and Gsc elevated, whereas Sox17 reduced upon Tcf7l1 ablation at days 2 and 4. g Ectoderm genes Pax6, Notch3, and Nestin showed increased expression upon Tcf7l1 ablation at several time points. Gene expression assayed by real-time RT-PCR. N ≥ 3; *p < 0.05 versus control cells. CMV cytomegalovirus, Ctr control, DAPI 4′,6-diamidino-2-phenylindole

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