Figure 3.

Dominant-negative BD1 of BRD4 inhibits CEBPD expression

(A) Diagram of the constructs used to exogenously express GFP (control) and a dominant-negative domain that competes with BD1 or BD2 of BRD4. (B and C) Effect of dominant-negative BD1 or BD2 of BRD4 on CEBPD expression. (D) Diagram to show selective binding of Olinone and RVX208 to BD1 and BD2, respectively, and binding of JQ1 to both; each of these bromodomain blockers binds all three BETs (BRD2, BRD3, BRD4). (E and F) Effect of bromodomain blockers on CEBPD expression. The data of the vehicle for Olinone and that for RVX208 (E) were pooled to generate the average vehicle value in the plot (black bar, F). MOVAS cells were transduced with lentivirus to express the GFP control or dominant-negative BD1 or BD2 for 24 h before harvest for western blot analysis. For pharmacological pretreatment, cells were incubated with vehicle (equal amount of DMSO) or a bromodomain blocker (10 μM RVX208 or 20 μM Olinone) for 4 h before harvest. Quantification: densitometry of western blots from independent repeat experiments was normalized to β-actin (similar band intensities on blots) and then averaged to calculate mean ± SEM; n = 3 independent repeat experiments. Statistics: one-way ANOVA followed by Bonferroni post hoc test; ∗p < 0.05, ∗∗p < 0.01; n.s., not significant.