The critical role of PRC1 in the chromosome clustering in the DNA hypomethylated ESCs.

(A): Representative immunostaining images of the surface-spread nuclei of the wild-type, Dnmt1-KO and Dnmt1/Tet1-DKO ESCs. The yellow squares indicate the enlarged areas shown on the bottom. The white dashed circles indicate the PCH. (B): Quantification of immunostaining of H2AK119ub, related to (A). The enrichment score is calculated by the relative signal intensity of PCH to the entire chromosome. An average of 3 chromosomes was calculated for each cell. n = 20 per cell line. (C): ChIP-qPCR for H2AK119ub at the major satellite repeats in the wild-type, Dnmt1-KO and Dnmt1/Tet1-DKO ESCs. Each bar represents relative enrichment to wild-type after normalization against H2A ChIP. n = 3 per cell line. (D): Representative immunostaining images of surface-spread nuclei of the wild-type, Dnmt1-KO and Dnmt1/Ring1A/1B-TKO ESCs. Cells were analyzed 4 d after the transfection of the CRISPR cassette. (E): Boxplot showing the number of distinct chromocenters in the nuclei of ESCs, related to (D). Wild-type, n = 97; Dnmt1-KO, n = 75; Dnmt1/Ring1A/1B-TKO, n = 64. P values were calculated using the Mann-Whitney U-test. ***P < 0.001; **P < 0.01. Scale bar, 5 μm.