BZ increases nuclear p65 accumulation, and IKK-dependent p65 recruitment to IL-8 promoter in TNBC cells.

<p><b>(A)</b> Western blotting of cytoplasmic (CE) and nuclear extracts (NE) prepared from MDA-MB-231 cells treated 24 h with BZ, and analyzed by using p65 and IκBα antibodies. The presence of cytoplasmic proteins in nuclear fraction was evaluated by re-probing the membrane with lactate dehydrogenase (LDH) antibody. Nuclear contamination in the cytoplasmic fraction was assessed using histone H3 specific antibody. To confirm equal protein loading, the membranes were stripped and re-probed with actin antibody. Each lane corresponds to approximately 5×10⁴ cells. <b>(B)</b> Densitometric evaluation of p65, IκBα, and histone H3 levels in nuclear extracts of BZ-treated MDA-MB-231 cells, shown in panel A. The densities of nuclear p65, IκBα, and histone H3 were normalized to the densities of nuclear actin. The values for untreated cells were arbitrarily set to 1, and the other values are presented relative to these values. <b>(C)</b> Recruitment of p65 to endogenous IL-8 promoter analyzed by ChIP and quantified by real time PCR in MDA-MB-231 cells treated with 100 nM BZ for 0, 3, 6 and 24 h. <b>(D)</b> ChIP of p65 recruitment to IL-8 promoter in MDA-MB-231 cells pre-incubated 12 h with control DMSO, Bay-117082 (10 μM), or SC-514 (10 μM), and then incubated 24 h with 100 BZ. The data are presented as the change in occupancy over the human IGX1A (Qiagen) sequence control and represent the mean +/− SE of three experiments. Asterisks denote a statistically significant change compared to control.