Figure 4

NF-κB binding to the ALCAM promoter and activity of the ALCAM promoter in breast cancer cell lines. A) p65 binding and trans-activation of the ALCAM promoter. (i) EMSA showing mobility of the -1140 NF-κB probe in the absence of nuclear extract (lane 1). Complexes B1, B2 and B3 form in the presence of LOX melanoma cell nuclear extracts (lane 2), subsequent lanes contain unlabelled wild-type NF-κB probe (lane 3), unlabelled mutant NF-κB probe (lane 4) and anti-p65 antibody (lane 5). (ii) ChIP assay showing PCR products for the -1140 ALCAM NF-κB motif and flanking DNA sequence amplified from chromatin of C8161.9 (lane1), LOX (lane 2) and Meljuso (lane3) cells precipitated with anti-p65 antibodies. Absence of PCR product in immunoprecipitation with non-immune IgG (lane 4), lane 5 (input DNA). (iii) Activity of p1200ALCAMLuc in C8161.9 and LOX cells over-expressing p65 or control vector. B) Schematic diagram of ALCAM reporter constructs and histogram showing relative luciferase activity for each in breast cancer cells with high (HCC70, MDA-MB-231, T47D), medium (BT549, CAMA-1) and low (HCC 1500, MCF-7,-SK-BR-3) ALCAM expression.