Fig. 5

Reduction of EZH2 impairs IDH2 dependent metabolic rewiring to suppress OC growth

(A) Representative images of EZH2 and IDH2 staining in OC tissues (n = 105). Scale bar: 100 μm

(B) IDH2 level was strongly positively correlated with EZH2 level in the OC cohort (n = 105). P value was determined by Pearson’s correlation test

(C) Kaplan-Meier survival analysis for the OC cohort (n = 105), which was divided into IDH2^high and IDH2^low subgroups based on the cutoff (cutoff = 5) calculated by the X-Tile software. The P value was calculated by log-rank test. **P < 0.01

(D) Colony formation assay in OVCAR8 and OVCAR4 cells transfected with IDH2 or EZH2 siRNA. Cells were cultured for 12 days. Growth medium was changed every 3 days. Images are representative of three independent experiments

(E) Cell growth curve of OVCAR8 and OVCAR4 cells transfected with IDH2 or EZH2 siRNA for 96 h. Data are presented as mean ± SD. *P < 0.05, **P < 0.01

(F-G) α-KG assay in OVCAR8 cells upon DNZep, GSK126 treatment (F) or in OVCAR8-shEZH2#2 cells ectopic expressing wildtype or mutant EZH2 (G). The OVCAR8 cells were treated with 2µM DZNep, 5µM GSK126 or 1 µg/mL doxycycline for 72 h before α-KG assay. Data are presented as the mean ± SD. **P < 0.01, ***P < 0.001

(H) Colony formation assay in OVCAR8 and OVCAR5 cells upon Dox-induced EZH2 silencing with or without α-KG supplementation. Cells were treated with 5ng/mL doxycycline for 12 days. Medium with or without 1mM α-KG was changed and doxycycline was also replenished every 2 days. Images are representative of three independent experiments

(I) Colony formation assay in OVCAR8-shEZH2#2 cells with ectopic IDH2 expression. Cells were treated with 5ng/mL doxycycline for 12 days. Growth medium was changed and doxycycline was also replenished every 3 days. Images are representative of three independent experiments. Immunoblot analysis of EZH2 and IDH2 levels in indicated samples. Cells were treated with or without Dox for 72 h before being harvested. GAPDH was used as loading controls

(J) Cell growth curve of OVCAR8-shEZH2#2 cells with ectopic IDH2 expression upon 1 µg/mL doxycycline treatment for 96 h. Data are presented as the mean ± SD. *P < 0.05

(K) Representative OCR pattern as a function of time (in min), normalized to total protein levels. Cells were incubated in the absence or presence of 1 µg/mL doxycycline for 72 h before OCR measurement. Data are shown as mean ± SD of three replicates per treatment