Figure 4: DZNep rescues p21 expression in hESCs, and p21 expression in hESCs induces differentiation.

(A,B) DZNep reduces H3K27me3 marks on the p21 locus without affecting p53 binding to the p21 promoter. H9 hESCs were treated with DZNep (1 μM) for 2 days and ChIP analysis was performed using p53 antibody (A) or H3K27me3 antibody (B) as in Fig. 3 (n = 3, mean ± SD). **p < 0.01, two-tailed unpaired t-test compared to control cells. The passage number of H9 hESCs is P35. (C) DZNep induces the expression of p21 in hESCs. H9 hESCs and hMECs were treated with DZNep at the indicated dose for 2 days. Proteins were harvested for Western blotting analysis with the indicated antibodies. The passage numbers of H9 hESCs and hMSCs are P35 and P9 respectively. (D) Ectopic p21 expression in hESCs. H9 hESCs were infected with retrovirus carrying either empty (pMSCV) or p21 and selected with blasticidin for 3 days. Immunofluorescence staining of p21 and differentiation marker, LMNA are shown. Scale bar, 20 μm. (E) Introduction of p21 reduces the expression of pluripotency markers and increases the differentiation markers in H9 hESCs. The mRNA expression of the indicated genes was analyzed as in Fig. 1C. The mean value of mRNA expression in pMSCV infected cells is set at 1, and relative expression is shown. **p < 0.01, ***p < 0.001, two-tailed unpaired t-test compared to control cells. (F) Expression of p21 in hESCs triggers a differentiated cell morphology (Phase) and reduces alkaline phosphatase staining (AP staining). Scale bar, 100 μm.