Early EMT and OGS induce the epigenetic changes (A) The enrichment of HIF1α, SNAI2, TWIST1/2, and ZEB1 binding sites on the promoters of Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 was determined by Pscan Zambelli et al, 2009().Figure 3(A) The enrichment of HIF1α, SNAI2, TWIST1/2, and ZEB1 binding sites on the promoters of Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 was determined by Pscan Zambelli et al, 2009(). The resulted Z-scores were listed. "Max. M" suggested the maximum Z-score generated with SNAI2, TWIST1/2, and ZEB1 binding sites on one indicated promoter. "Average" suggested the average Z-score of all transcriptional factors tested in the Pscan software on one indicated promoter. The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with mES (B) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups. The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with 5C medium (C). The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups. pre-iPSCs were isolated during reprogramming with mES-Vc, mES or 5C medium. These pre-iPSCs were further cultured with a different medium (mES-Vc and mES medium) for seven days. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. pre-iPSCs were isolated during reprogramming with mES-Vc, mES or 5C medium. These pre-iPSCs were further cultured with a different medium (mES-Vc and mES medium) for seven days. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The numbers of Oct4GFP+ colonies were determined on day 7 (E). Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (G). Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. In addition, energy metabolism was determined on day 6 with Seahorse instrument Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. In addition, energy metabolism was determined on day 6 with Seahorse instrument Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. Cell migration was determined by measuring the migration rate with live-cell imaging on day 6 (J). Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was determined on day 6 with qPCR (K). (L) Early EMT and facilitated OGS cooperate to induce pluripotency by removing the additional epigenetic barrier.