Fig. 3.

MUC1-C→NF-κB p65 signaling induces BCL2A1 expression. a Lysates from MDA-MB-468/tet-MUCshRNA cells treated with or without DOX for 7 d were immunoblotted with the indicated antibodies. b Schema of the BCL2A1 promoter with localization of the NF-κB binding motif at −736 to −726 upstream of the transcription start site (TSS). c MDA-MB-468 cells were stably transduced to express control shRNA (CshRNA) or NF-κB p65 shRNA (p65shRNA). Cells were analyzed for BCL2A1 mRNA levels by qRT–PCR. The results (mean ± SD of 3 determinations) are expressed as BCL2A1 mRNA levels relative to those in CshRNA cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right). d MDA-MB-468 cells treated with 5 μM BAY-11-7085 or vehicle control for 30 h were analyzed for BCL2A1 mRNA levels by qRT–PCR. The results (mean ± SD of three determinations) are expressed as BCL2A1 mRNA levels relative to those in control cells (assigned a value of 1) (left). Cell lysates were immunoblotted with the indicated antibodies (right). e Soluble chromatin from MDA-MB-468/tet-MUC1shRNA cells cultured with or without DOX for 5 d was precipitated with anti-NF-κB p65 or a control IgG. The final DNA samples were amplified by qPCR with primers targeting the NF-κB binding region in the BCL2A1 promoter. The results (mean ± SD of three determinations) are expressed as the relative fold enrichment compared with the IgG control (assigned a value of 1)