Figure 1: p21 expression is suppressed in human embryonic stem cells.

(A) p21 expression is suppressed in hESCs and hiPSCs compared to hMSCs. Protein lysates from the indicated cells were analyzed by Western blotting with the indicated antibodies. The passage number is shown in brackets. 70 μg of protein lysate was loaded in each lane. (B) p21 expression in hESCs is about 50 times lower than in hMSCs, as analyzed by Western blotting with the indicated antibody. 150 μg of protein lysate from H9 hESCs was loaded in lane 1. The amount of total protein lysate loaded relative to hESC is indicated. (C) p21 mRNA levels are lower in H9 hESCs than in hMSCs, as assessed by qRT-PCR (n = 3, means ± SD). The mean value of mRNA expression in H9 hESCs is set at 1, and relative expression is shown. TBP was used as an internal control for normalization. (D,E) p53 is required for p21 expression in H9 hMSCs (passage number 8). H9 hMSCs were transfected with control and p53 siRNAs. p21 protein levels (D) were analyzed by Western blotting. 50 μg of protein lysate was loaded in each lane. p21 mRNA levels (E) were analyzed as in (C). The mean value of mRNA expression in control siRNA transfected cells is set at 1, and relative expression is shown. (F,G) p21 expression in H9 hESCs remains very low upon p53 activation by DNA damage. H9 hESCs and H9 hMSCs were treated with the indicated concentration of etoposide (F) or hydroxyurea (G) for 24 hrs and harvested for Western blotting. The passage numbers of H9 hESCs and hMSCs are P37 and P10 respectively. 50 μg of protein lysate was loaded in each lane.