Figure 3.

Foxo1 and Foxo3 regulate pluripotency of mESCs. (a) Alkaline phosphatase staining in Foxo1-, Foxo3- or control-knockdown mESCs. Scale bar, 50 μm. (b,c) qRT-PCR analysis of pluripotency (top) and developmental genes (bottom) in mESCs expressing one of two distinct shRNA targeting Foxo1 (b) or Foxo3 (c). shRNA-mediated knockdown of luciferase was used as a control. The gene expression levels of four lineages, including trophectoderm (Cdx2 and Mash2, also known as Ascl2), endoderm (Gata6 and Foxa2), ectoderm (Cxcl12, Mash1, also known as Ascl1, and Fgf5) and mesoderm (brachyury) were examined after knockdown of Foxo1 or Foxo3. All graphs show mean ± s.e.m. for n = 3; *P < 0.05, **P < 0.01. P values are from comparing Foxo1 shRNA 4 or Foxo3 shRNA 4 with Luc shRNA 1 (b,c). Ectopic expression of a resistant form of the targeted FoxO1-rescued FoxO1-knockdown-mediated phenotype in both hESCs and mESCs. (d) hESCs were transfected with a lentivirus expressing FOXO1 shRNA III (targeting the 3′ untranslated region of the FOXO1 mRNA); GFP-positive cells were FACS sorted 3 days later (>50-60% GFP positive), transduced with lentiviral vector (pLEIGW-FOXO1) that contains only the FOXO1 coding region and cultured for another 2 days before gene expression analysis. All results are relative to the endogenous ACTB. Error bars indicate s.e.m. (n = 3). *P < 0.05. (e) Endogenous Foxo1 was targeted by Foxo1 shRNA 4 in mESCs and rescued by re-expressing the shRNA-resistant Foxo1-m4 construct (EF1-Foxo1-m4). EF1 promoter empty vector (EF1) was used as a control. Gene expression analysis of Foxo1 and pluripotency genes was carried out by qRT-PCR and all results in Foxo1-shRNA 4 cells are expressed relative to the sample with Luc shRNA and EF1 (set as 1). Error bars indicate s.e.m. (n = 3). P < 0.05 for all genes in rescued samples when compared with controls.