Fig. 5

BRD protein knockdown effect on ovarian cancer BET-associated mechanisms. A siRNA-mediated knockdown in OVCAR-3 cells resulted in significant BRD2, BRD3 and BRD4 transcript down-regulation after 48 h compared to the control treatment (siNeg). B Protein lysates of OVCAR-3 cells treated with specific siRNAs were subjected to western blot analyses to study changes in BRD2, BRD3, BRD4, p21, cleaved PARP (cPARP) and γH2A.X protein levels after 48 h of knockdown (KD). The right panel displays proportional differences between relative densities of BRD proteins in KD and control samples calculated using ImageJ. C Protein lysates of OVCAR-3 cells treated with different combinations of siRNAs were subjected to western blot analyses. The two controls correspond to different amounts of scrambled siRNA introduced in cells to mimic the action of 2 or 3 target siRNAs (Ctrl 2 and Ctrl 3). D Cell lysates from OVCAR-3 cells treated with combinations of siRNAs (2, 3 and 4) for 48 h were subjected to qRT-PCR to study mRNA expression changes of i-BET858 targets NRG1, CCR1 and LIF. Each KD was compared to their correspondent control sample which included different amounts of scrambled siRNA; only one control was plotted to simplify (siNeg). E Flow cytometry apoptosis analysis of OVCAR-3 cells treated with siRNA targeting BRD2, BRD3 and BRD4 for 48 h. Cells were stained with propidium iodide and Annexin V-FITC rendering 4 populations: viable (−, −), early apoptotic (−, +), late apoptotic (+, +) and dead (+, −). Graphs display cell densities, whereby red, green and blue colours indicate high, medium and low cell densities, respectively. F Flow cytometry cell cycle analyses of OVCAR-3 cells treated with siRNA targeting BRD2, BRD3 and BRD4 for 48 h. Blue peaks represent cells in G0/G1 phase, whilst green peaks represent cells in G2/M phase. The area depicted as yellow represents cells in S phase. All values represent the mean ± SD of three biological samples (*P < 0.05, ***P < 0.001, ****P < 0.0001)