Figure 4.

Differentiation Induced by Blocking of PLK4 or STIL Is p53 Dependent

Cells were transfected with either control or p53 siRNA, or the expression of p53 was permanently downregulated by CRISPR/Cas9 (p53 low cells) and subsequently treated as indicated.

(A) Analyses of mRNA levels of TP53 after siRNA transfection in hESCs, showing the efficiency of p53 knockdown. Data are presented as relative fold change over control.

(B) Phase-contrast images of hESCs following siRNA transfection and 2 days of treatment; black arrows indicate mitotic cells. Scale bars, 50 μm.

(C) Number of cells in described conditions was measured at indicated time points by crystal violet assay and plotted as growth curves. First panel shows siRNA data (n = 1), second panel shows analyses of p53 low hESCs and their respective controls (n = 3), and third panel shows p53 low hiPSCs (n = 3).

(D) Expression of differentiation markers (T and PAX6) after siRNA transfection and 4 days of treatment in hESCs, analyzed by qPCR. Data are presented as relative fold change over control (first column).

(E–G) Western blot analyses of rescue of the centrinone treatment-induced effects by p53 downregulation either by siRNA (2 days of treatment, E) or CRISPR/Cas9 (p53 low hESCs/hiPSCs; 3 days of treatment, F and G). Samples were probed with indicated antibodies, with actin as a loading control.

Data are presented as mean ± SEM (^∗∗p < 0.005, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001).