Skip to main content
. Author manuscript; available in PMC: 2022 Nov 16.
Published in final edited form as: Cancer Res. 2022 May 16;82(10):2019–2030. doi: 10.1158/0008-5472.CAN-21-0871

Figure 4. EZH2 loss stimulates the AKT pathway through an epigenetic switch that up-regulates EPHA2.

Figure 4

(A) WB analyzing the expression of indicated proteins in EZH2-WT and EZH2-KO #51 and #68 medulloblastoma cells. (B) WB analysis of indicated proteins in EZH2-WT and EZH2-KO #51 medulloblastoma cells that were mock-treated or treated with 10μM CBP/p300 inhibitor C646 for 24h. (C) MTT assay showing percentage of viable cells in EZH2-WT and EZH2-KO #51 medulloblastoma cells. Cells were mock-treated or pre-treated with 10μM CBP/p300 inhibitor C646 for 24h and either mock-irradiated or exposed to indicated doses of IR. Percentage of viable cells was determined 7d after IR. (D) ChIP-Seq analysis of the H3K27me3 and H3K27ac levels within the indicated genomic loci in EZH2-WT and EZH2-KO #51 medulloblastoma cells. (E) ChIP analysis of H3K27me3 and H3K27ac levels within the Epha2 (left panel) and Pik3cb (right panel) gene promotors in EZH2-WT and EZH2-KO medulloblastoma cells. (F) qRT-PCR analysis showing the transcriptional activity of indicated genes in EZH2-WT and EZH2-KO #51 medulloblastoma cells. (G) WB analysis of indicated proteins in EZH2-WT and EZH2-KO #51 medulloblastoma cells. (H) Images of IHC staining of EPHA2 in H3K27me3-deficient S17, S24 (left panel) and H3K27me3-proficient S10, S13 (right panel) tumor samples from non-WNT/SHH medulloblastoma patients. (I) WB analysis of indicated proteins in EZH2-WT and EZH2-KO #51 medulloblastoma cells that were mock-treated or pre-treated with indicated concentrations of the EPHA2 inhibitor ALW-II-4127 for 24h followed by exposure to 4Gy IR. Cells were harvested for WB analysis 2h following IR. (J) MTT assay showing percentage of viable cells in EZH2-WT and EZH2-KO #51 medulloblastoma cells. Cells were pre-treated with 100nM EPHA2 inhibitor ALW-II-4127 for 24h and either mock-irradiated or exposed to 4Gy IR. Percentage of viable cells was determined 7d after IR. (C, F, J) Data of three independent experiments are shown as mean +/− SD. Paired two-tailed T-test was used for statistical analysis (***p < 0.001; **p < 0.01; *p < 0.05). (E) Mean of two independent experiments is shown.