Fig. 2

EGF stimulates binding of NF-κB to the promoter region of FOXC1. a MDA-MB-468 cells were serum-starved overnight and then treated with EGF for 24 h. FOXC1, p65, p50 levels in nuclear and cytoplasmic fractions were examined using immunoblotting. Lamin A/C was used as a nuclear marker and actin was used as a cytoplasmic marker. b Two conserved putative NF-κB binding sites (underlined; −1856 to −1877 and −1698 to −1719) in the cloned FOXC1 promoter. NF-κB probe sites are highlighted in red. c MDA-MB-468 cells were serum-starved overnight and treated with EGF for 24 h in the presence or absence of 10 μM Bay 11–7082 (Bay; NF-κB inhibitor) after preincubation with the inhibitor for 1 h. Nuclear protein was extracted. EMSA analysis was conducted using biotin-labeled double-stranded NF-κB probes. d MDA-MB-468 cells were serum-starved overnight and treated with EGF for 24 h. Nuclear protein was extracted and mixed with biotin-labeled double stranded NF-κB probes and streptavidin beads. p65-probe interaction was examined with immunoblotting. e Top, serum-starved MDA-MB-468 cells were treated with or without EGF for 24 h and fixed by formaldehyde. ChIP assays were performed using p65 antibody to examine the binding of p65 to the FOXC1 promoter. The PCR amplified FOXC1 promoter region is indicated by solid arrow (see the diagram in B). Bottom, MDA-MB-468 cells were treated with EGF for 24 h after preincubation with the NF-κB inhibitors for 1 h: 5 μM BMS-345541 (BMS; IKK inhibitor III), 10 μM Bay 11–7082 (Bay; NF-κB Activation Inhibitor II) and 50 μM JSH-23 (JSH; NF-κB Activation Inhibitor II). Then ChIP assays were performed. f The insert shows schematic diagrams of the two putative NF-κB binding sites in the FOXC1 promoter in which the two NF-κB binding sites were mutated by site-directed mutagenesis (see Materials and Methods). MDA-MB-468 cells were transfected with the wild-type or mutated FOXC1 promoter and NF-κB constructs. Cells were treated with EGF or vehicle for 24 h, followed by luciferase assays