Fig. 7.

Effects of Sox9 on early ESC differentiation requires p21. (A) Western blots detecting P21 and β-actin (control) in SOX9-inducible ESCs cultured for 4 days in Dox+ (control) or Dox− conditions after treating with siRNA against P21 or control siRNA. (B) qPCR analyses showing the expression of P21 and β-actin (control) in SOX9-inducible ESCs cultured in Dox+ (control) or Dox− conditions. (C,F,I,L) Immunostaining of SOX9-inducible ESCs cultured for 4 days in Dox+ (control) or Dox− conditions after treating with siRNAs against P21 or control siRNA: mesoderm marker (T), endoderm marker (SOX17), ectoderm marker (NES) and pluripotency marker (SOX2). Scale bars: 200 µm. (D,G,J,M) Examples of FACS analyses: SOX9-inducible ESCs cultured for 4 days (D,G,M) or 6 days (J) after treating with siRNA against P21 or control siRNA, and stained for FLK1⁺, FOXA2⁺ or SSEA1⁺. (E,H,K,N) A summary of three independent FACS experiments (data are mean±s.e.m.; *P<0.05 versus Dox+). (O) ChIP assays examining the binding of SOX9 to the UTR region and SRR2 enhancer region of SOX2, and to the Nanog promoter region in SOX9-inducible ESCs cultured for 3 days after treating with siRNAs against P21 or control siRNA (three independent experiments, data are mean±s.e.m.; *P<0.05 versus Dox+). (P) A schematic diagram showing molecular interactions that regulate the differentiation of ESCs into three germ layers: Sox9 induces p21, which then inhibits Sox2 expression through its direct binding to the SRR2 enhancer of Sox2 gene. Subsequently, other pluripotency genes, such as Pou5f1 and Nanog, are downregulated, which accelerates the differentiation of ESCs into three germ layers.