Fig 2: Effect of high glucose on Ezh2 recruitment at the MMP-9 promoter. The binding of Ezh2 at the MMP-9 promoter was quantified using Ezh2 monoclonal antibody, (a) followed by amplification of the promoter region by real time qPCR. (b) Product sizes were confirmed on 2% agarose gel. *P < 0.05 compared with 5-mM glucose; #P < 0.05 compared with 20-mM glucose.

Fig 3: Effect of regulation of Ezh2 on MMP-9: (a) mRNA levels of MMP-9 were measured using real time qPCR, and each measurement was made in duplicate in three to five samples in each group. (b) Enzyme activity of MMP-9 was quantified in by an ELISA method using a fluorescence kit. (c) Mitochondrial localization of MMP-9 was determined by immunofluorescence using DyLight 488-conjugated secondary antibody for MMP-9 and Texas red-conjugated for CoxIV. *P < 0.05 compared with 5-mM glucose; #P < 0.05 compared with 20-mM glucose.

Fig 4: Effect of CCNDBP1 on the changes in various proteins in HCC cells after X-ray irradiation. (a) Western blotting of proteins related with the ATM–CHK2 pathway in the cells harvested at the indicated times after X-ray irradiation (immediately and at 24, 48, and 72 h). (b) The relative expression ratios of the proteins are shown. The values represent mean ± standard deviations (n = 5), * p < 0.05, ** p < 0.01 on one-way analysis of variance followed by Bonferroni’s multiple comparison test. CCNDBP1, cyclin d1 binding protein 1; EZH2, enhancer of zeste homolog 2; ATM, ataxia telangiectasia mutated; pATM, phosphorylated ataxia telangiectasia mutated; CHK2, checkpoint kinase 2; pCHK2, phosphorylated checkpoint kinase 2; CDC25C, cell division cycle 25 homolog C.

Fig 5: The effect of EZH2 on PAR4-activated ß-catenin half-life. (A) Western blot analysis of ß-catenin in HEK293 cells co-transfected with 0.4 µg hPar4, 0.3 µg flag-ß-catenin and 0.5 µg ezh2-HA plasmids and activated by PAR4 with 200 µM AYPGKF for 2 h followed by cycloheximide treatment (100 µg/mL) for between 5 min and 2 h. Western blot analysis was performed on cell lysates to detect ß-catenin using anti-flag antibody (1:1000) and normalized to ß-actin (1:1000) for protein loading. (B) HEK293 cells were co-transfected with 0.075 µg TOPflash 0.125 µg LEF, 0.2 µg ß-gal, 0.2 µg Par4, and 0.1 µg ezh2-HA. Following activation with 200 µM AYPGKF and treatment with cycloheximide, lysates were collected, and luciferase activity was normalized to ß-gal activity to control for transfection efficiency. This experiment was carried out in triplicate.