Figure 1.

P53 Is Essential for the Elongated G1 Phase in 2i ESCs

(A) Western blot (WB) of P53 level in total cell lysate of WT serum and 2i ESCs.

(B and C) (B) Proteomics analysis and WB from different cellular fractions showing a higher level of chromatin-associated P53 protein in 2i ESCs compared with serum ESCs. Data from three biological replicates; significance was tested using an unpaired t test. (C) Cells in G1 phase expressing the FUCCI reporter are Kusabira Orange-positive, and cells in G2 phase are Azami Green-positive. Schematic representation of CRISPR/Cas9-mediated P53 knockout in FUCCI reporter ESCs targeting the common exon in Trp53 isoforms, resulting in the absence of P53 as shown in the WB (P53^−/− clone 1).

(D) Three independent P53^−/− clones were obtained, all showing a significant decrease in the expression of P21.

(E) Analysis of FUCCI reporter expression in WT and P53^−/− cells. The longer G1 phase in 2i conditions (when compared with serum) is abbreviated in P53^−/− 2i ESCs, whereas in serum ESCs, no differences between WT and P53^−/− ESCs are observed. Reporter expression in P53^−/− clone 1 is shown (experiment performed in triplicate), which is representative for the three independent P53^−/− clones in at least two independent experiments.

(F) Quantification of the different phases of the cell cycle in WT and P53^−/− (clone 1) FUCCI cells using BrdU incorporation combined with PI staining. Data from an experiment performed in triplicate that is representative for two independent clones.

(G) BrdU/PI staining on WT and P53^−/− EB5 cells confirms the decrease in the percentage of G1-phase cells upon deletion of P53 in 2i conditions. Numbers indicate mean plus SD from a technical replicate.

(H) WB showing decreased expression of P21 during G1 phase in P53^−/− FUCCI ESCs in 2i (P53^−/− clone 1).

(I) RT-qPCR reveals a reduction in Cdkn1a mRNA levels in P53^−/− (clone 1) FUCCI ESCs and in the independent P53^−/− EB5 ESC line when compared with WT ESCs. No decrease in Cdkn1b mRNA was observed. Data from three technical replicates for each cell line are shown (bar graph shows mean and standard deviation).

(J) Volcano plot showing transcriptome changes in P21^−/− G1-phase ESCs compared with the WT G1-phase ESCs cultured in 2i conditions. Each dot represents one gene. Significantly changed genes (adjusted p value <0.1 and a fold change of >1.5) are colored (downregulated genes in green and upregulated genes in red). GO clusters show the biological processes significantly enriched among the differential genes.