Figure 4.

Dual inhibition of enhancer of zeste homolog 1/2 (EZH1/2) overactivates WNT signaling by reducing the level of trimethylated histone H3 at lysine 27 (H3K27me3). A, qRT‐PCR analysis showing relative expression of WNT‐related genes in MM.1S and RPMI8226 cells treated with 1 μmol/L GSK126 or OR‐S1 for 72 h. Y‐axis represents the fold‐change in gene expression after normalization to that of ACTB. Error bars represent the mean ± SD. B, Western blot analysis of non‐phosphorylated (active) β‐catenin in MM.1S and RPMI8226 cells treated with DMSO or 1 μmol/L OR‐S1 for 72 h. α‐Tubulin was used as a loading control. C, ChIP‐qPCR of H3K27me3 at the loci of WNT‐related genes in MM.1S and RPMI8226 cells treated with 1 μmol/L OR‐S1 for 72 h. Level of H3K27me3 was normalized against that of total H3. Background signals in each sample were evaluated by ChIP using rabbit IgG (green). CCND1 and ACTB loci were used as positive and negative controls, respectively. Error bars represent the mean ± SD. *P < .05; **P < .01; ***P < .0001 (Student's t test). N.D., not detected