p53 Regulates Cell Cycle and MicroRNAs to Promote Differentiation of Human Embryonic Stem Cells
Figure 4
p53 drives differentiation of hESCs.
(A) AP staining. hESCs transfected with non-target (siControl) or siRNA specific to p53 (siTP53) or p21 (siCDKN1A) were treated with RA and stained for AP (blue colonies). (B and C) hESCs transfected and treated as in (A) were used in Western blotting (B) and qRT-PCR (C) analyses. The blots in (B) were quantitated, and average density of three different blots is plotted (bottom panel) (*, p<0.05) (mean ± SEM). (D) OCT4 + SSEA4 staining. hESCs transfected with siRNA followed by RA treatment were stained for SSEA4 and OCT4 and subjected to dual flow cytometry analysis. Triplicate samples were analyzed in each experiment, and data analyzed with FACSDiva software. Decreases in fraction of OCT4-positive cells, as compared to siControl untreated, are indicated in red. (E) AP staining. hESCs transfected with HDM2 or TRIM24 siRNA were treated with RA and stained for AP. (F) Quantified AP-stained colonies. Date shown are for 50 colonies per treatment in three separate experiments (in [A] and [E]), scored as undifferentiated, partially differentiated, or fully differentiated colonies, mean ± SEM. (G) OCT4 + SSEA4 staining and flow cytometry analysis as in (D) after transfection with siRNA targeting HDM2 or TRIM24. (H) Cell cycle analysis. hESCs transfected with HDM2 or TRIM24 siRNA were stained with PI and subjected to cell cycle analysis. (Also see Figures S3 and S5.).