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Fig. 2 | Molecular Cancer

Fig. 2

From: EZH2 mediated metabolic rewiring promotes tumor growth independently of histone methyltransferase activity in ovarian cancer

Fig. 2

EZH2 functions as an oncogenic driver to promote OC growth independently of its catalytic activity

(A) Colony formation assay in three OC cell lines. Cells were treated with DMSO or four different EZH2 inhibitors with indicated concentrations for 12 days. Growth medium was changed and inhibitors were also replenished every 3 days. Images are representative of three independent experiments

(B) Cell growth curve of 3 OC cell lines treated with DZNep (1µM), YM281 (5µM), GSK126 (5µM) or EPZ-6438 (5µM) for 96 h. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

(C) Immunoblotting analysis of EZH2 and H3K27me3 in 3 OC cell lines treated with four different EZH2 inhibitors with indicated concentrations

(D) Tumorsphere formation assay of OC cells treated with DZNep (1µM), YM281 (5µM), GSK126 (5µM) for 10–12 days. Data are presented as the mean ± SD. **P < 0.01, ***P < 0.001

(E-F) Xenograft tumor growth (E) and tumor weight (F) of OVCAR8 cells in nude mice treated with saline as control, EPZ-6438 at 200 mg/kg intragastrically daily, or DZNep at 1 mg/kg intraperitoneally twice a week. n = 7 per group. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001(Two-way ANOVA with Tukey’s post hoc test)

(G) IHC analysis of EZH2, H3K27me3, and Ki67 levels in the excised tumors from (F). Data are presented as mean ± SEM. ***P < 0.001

(H) Immunoblot analysis of EZH2 and H3K27me3 levels in shEZH2-inducible OVCAR8 cell lines with wildtype or mutant EZH2 restoration. Expression of EZH2-WT and the EZH2-ΔSET was detected by an EZH2 antibody against residues surrounding Arg354 of human EZH2 protein (SET domain locates in 612–727 amino acids). GAPDH and Histone H3 were used as loading controls

(I) Cell growth curve of shEZH2-inducible OVCAR8 cell lines with wildtype or mutant EZH2 restoration in the presence or absence of Dox at 1 µg/mL for 96 h. Data are presented as mean ± SD. ***P < 0.001

(J) Colony formation assay in shEZH2-inducible OVCAR8 cell lines with wildtype or mutant EZH2 restoration in the presence or absence of 10ng/mL Dox for 12 days. Growth medium was changed and doxycycline was also replenished every 3 days. Images are representative of three independent experiments

(K) Tumorsphere formation assay of indicated cells treated with or without Dox at 10ng/mL for 10 days. Data are presented as the mean ± SD. **P < 0.01, ***P < 0.001 vs. EV groups

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