Fig 1: CBX5 is identified as a repression target of BRD4(A) Differentially regulated genes in Ovcar8 cells were identified upon BRD4 knockdown and chemical inhibition by JQ1. The number of entities are shown with different cut-offs; numbers in bold are the entities used in B. (B) Up- and down-regulated genes are compared separately. (C) Candidate genes were validated by qPCR in Ovcar8 cells. Blue bar represents the average of 3 independent biological replicates of JQ1 treatment and red bar shows results from the siRNA transfection, and green bars represent serum starvation, as an experimental condition for non-specific cell cycle arrest. Each gene was normalized by GAPDH and compared to the negative control for each condition (starved/unstarved, siBRD4/siNEG, JQ1/DMSO). *, p<0.05 in ANOVA with Dunnet post-hoc correction for comparison of each to GAPDH. (D) Up-regulation of CBX5 and HIST1H2BD genes was validated in 3 more OC cell lines upon BRD4 knockdown. Error bars represent standard error of the mean. ANOVA with Dunnett post-hoc showed all p-values <0.05. (E) Relative proportion of cells in each phase of the cell cycle was measured with propidium iodide under conditions of JQ1 treatment, serum starvation, or BRD4 knock down. Error bars represent standard error of the mean. (F) Ovcar8 cells were transfected with indicated siRNAs. Total protein lysates (30µg) or nuclear extracts (20µg) were analyzed by Western blot. GAPDH and H2B were loading controls. (G) Nuclear extracts were probed for changes after BRD4 knockdown, using H2B as a loading control. (H) Cells were treated with JQ1 (2.5 µM) for 24hr; total protein was analyzed by Western blot with GAPDH as loading control.