Figures & data
Figure 1. Inhibition of EZH2 decreases BRCA1 mRNA level but increases BRCA1 protein level. (A) The mRNA level of BRCA1 was decreased in shEZH2 transfected cells compared with untransfected ES2, SKOV3, and A2780 cells. (B) Immunoblot assays revealed that total protein expression of BRCA1 was increased in stable transfected shEZH2-A2780 cells. (C) Left, loss of EZH2 increased BRCA1 nuclear protein level, but decreased BRCA1 cytoplasmic protein level. N and C represent nuclear and cytoplasmic protein respectively. Right, quantization of western blot. (D) Immunocytochemistry analysis shown that stable inhibition of EZH2 in A2780 cells increased BRCA1 protein expression and induced BRCA1 nuclear localization (×400).
Figure 2. Depletion of BRCA1 partially rescues the biological effects of EZH2 inhibition in ovarian cancer. (A and B) The efficiencies of siBRCA1s silencing in A2780 cells were detected at mRNA (A) and protein (B) levels by qRT-PCR and western blotting respectively. (C) The shEZH2-mediated BRCA1 overexpression was abolished by cotransfection of siBRCA1-2 or -3. (D) Quantization of western blot. (E and F) Cell proliferation and cell cycle analyses were detected by MTT assay and flow cytometry respectively; showing that cotransfection of siBRCA1 partly rescued the reduction in cell proliferation and depressed the elevated percentage of G2/M phase caused by shEZH2 transfection in A2780 and ES2 cells. (G) Transwell migration assays revealed that inhibition of BRCA1 inversed the suppression in cell migration in A2780 and ES2 cells with shEZH2 transfection (×200).
Figure 3. EZH2-induced BRCA1 nuclear/cytoplasmic shuttling requires the activation of Akt-1. (A) Western blot analyses showed that the protein level of p-Akt-1(Ser473) was decreased by downregulation of EZH2 and inceased by IGF-1 treatment. (B) Immunocytochemistry analyses showed that activation of Akt-1 inversed EZH2-mediated BRCA1 nuclear/cytoplasmic shuttling (×400).
Figure 4. Interaction of EZH2 and BRCA1 in acquired cisplatin-resistant ovarian cancer cells. (A and B) The elevated mRNA (A) and protein (B) expression of EZH2 and BRCA1 were detected by qRT-PCR and western blotting respectively. (C) QRT-PCR shown the mRNA levels of EZH2 and BRCA1 in A2780/DDP cells with shEZH2 and siBRCA1 transfection alone or simultaneously. (D) Left, western blotting shown the protein expression of EZH2 and BRCA1 in cells with shEZH2 and siBRCA1 transfection independently or simultaneously. Right, quantization of western blot. (E) Following cisplatin treatment, MTT assays assessed the survival rates of cells with different expression of EZH2 and BRCA1.
