Fig. 4

Otx2 controls Hmga2 expression in epiblast-like stem cells (EpiLCs). a Phenotypic analysis of Otx2 knockout (KO) and wildtype (wt) differentiated embryonic stem cells (ESCs). The indicated markers were analyzed by qPCR at 4 days of differentiation as serum-free embryoid bodies (SFEBs) or into EpiLCs. qPCR data represent the means of independent biological replicates (Oct4, n = 4; Nanog, Rex1, and Pax6, n = 3; Klf4, n = 5) ± SEM. *P <0.05, **P <0.01. b Western blot analysis of undifferentiated and differentiated ESCs. Expression levels of Hmga2, Oct4, and Otx2 were measured in undifferentiated ESCs, during neuroectodermal differentiation through SFEBs and in EpiLCs. c Expression profile of Otx2, Oct4, and Hmga2 mRNAs at the indicated time points during ESC differentiation analyzed by qPCR. Data represent mean ± SD from two independent biological replicates (n = 2). d Otx2 mRNA levels in undifferentiated ESCs and in SFEBs at day 4 transfected with control or Hmga2 siRNAs. Data represent mean ± SD from two independent biological replicates (n = 2). e Oct4 and Otx2 expression in SFEBs at 2 and 4 days of differentiation derived from ESCs transfected with the indicated siRNAs. f Wt and Otx2 KO ESCs were induced to differentiate into SFEBs for 4 days. The expression of Hmga2 was analyzed by qPCR. Data represent mean of three independent experiments (n = 3) ± SEM. **P <0.01. g Hmga2 protein levels were measured in wt and Otx2 KO undifferentiated ESCs and 4-day differentiated SFEBs. h Otx2 association with the genomic region upstream Hmga2. ChIP-qPCR analysis of Otx2 binding to the genomic region upstream the transcriptional start site of Hmga2 (–9 kb) in EpiLCs. qPCR data represent mean of four independent experiments (n = 4) ± SEM