Figure 3
Downregulation of CDK1 leads to the loss of pluripotency, differentiation and polyploidy. (a) Expression analysis of main pluripotency genes by quantitative RT-PCR. Results are presented as average±S.E.M. (n=3). The value for the hESC transfected with control siRNA was set to 1 and all other values were calculated with respect to that. T-test analysis was carried out to assess the differences in gene expression between the control and CDK1 siRNA group, *P<0.05. (b) Upregulation of differentiation markers analysed by quantitative RT-PCR. Results are presented as mean±S.E.M. (n=3). The value for the hESC transfected with control siRNA was set to 1 and all other values were calculated with respect to that. T-test analysis was carried out to assess the differences in gene expression between the control and CDK1 siRNA group, *P<0.05. (c) Western blot analysis for the expression of key pluripotency markers. Note that the protein level of KLF4 and LIN28 was not restored by day 4 post transfection. GAPDH was used as a loading control. Data are representative of at least three independent experiments. (d) Alkaline-phosphatase-positive staining was observed in hESC transfected with control siRNA (upper row) but differentiated morphology and lack of typical staining was observed in cells transfected with CDK1 siRNAs (lower panel), 48 h post transfection. Images are representative of at least three independent experiments. (e) Representative histogram showing the percentage of polyploid cells in the CDK1 siRNA group at 48 h post transfection. At least 300 cells were analysed in each experiment. (f) Examples of appearance of multinucleated cells (black arrows) on a second day after CDK1siRNA treatment. Scale bar=50 μm
