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. Author manuscript; available in PMC: 2014 Jun 11.
Published in final edited form as: Nat Cell Biol. 2011 Jul 31;13(9):1092–1099. doi: 10.1038/ncb2293

Figure 2.

Figure 2

Reversible effect of loss of FOXO1 on pluripotency and differentiation of hESCs. (a) qRT-PCR analysis of pluripotency (left), mesoderm (middle) and endoderm (right) gene expression in H1/FOXO1-shRNA hESCs. Cells were maintained under pluripotency self-renewing conditions and treated with or without doxycycline for up to 4 days, after which cells were washed extensively and maintained in the absence of doxycycline. Quantification of the genes was relative to the endogenous β-actin transcript levels. Error bars indicate s.e.m. of three independent experiments, each carried out in triplicate. *P <0.05, *P <0.01. (b) In H1 cells, ectopic expression of FOXO1 and not FOXO3A induces expression of pluripotency genes. GFP-positive hESCs lentivirally transduced with an empty control vector or a vector containing FOXO1 or FOXO3A were FACS sorted 72 h after transduction and analysed by qRT-PCR for FOXO1 or FOXO3A and pluripotency marker gene expression; results shown are relative to endogenous ACTB and normalized to untransduced H1 cells under self-renewal conditions. Error bars indicate s.e.m. of three independent experiments each, carried out in triplicate; *P < 0.05, *P < 0.01, *P < 0.001. NT, not transduced. NS, not significant. (c) FOXO1-knockdown-mediated loss of pluripotency and induction of differentiation markers in H1/FOXO1-shRNA cells after several passages. Morphology (left) and alkaline phosphatase staining (right) of hESCs cultured in doxycycline for five passages. (d) Expression of surface markers of pluripotency, SSEA4, TRA-1-60 and TRA-1-81, by immunostaining and DAPI counterstaining of hESCs was analysed after five passages in the absence or presence of doxycycline (Dox). Scale bar, 100 μm. One representative of two independent experiments is shown. (e) qRT-PCR analysis of FOXO1 and pluripotency genes (left), mesoderm (middle) and endoderm (right) markers in H1 cells maintained in pluripotency self-renewal conditions with or without doxycycline for five passages. Results shown are relative to the endogenous ACTB. Error bars indicate s.e.m. (n = 3). *P < 0.05, ***P < 0.001.