Fig.5

Sp1 is involved in the up-regulation of Epha3 in RAGE-overexpressing BC cells. A mRNA expression of EPHA3 in RAGE-overexpressing (MCF7/RAGE and T47D/RAGE) respect to wild type (MCF7/wt and T47D/wt) cells, as ascertained by real-time PCR. Values are normalized to the actin beta (ACTB) expression and shown as fold changes of mRNA expression in RAGE-overexpressing respect to wild type cells. B-C Immunoblots of EphA3 in wild type (MCF7/wt and T47D/wt) and RAGE-overexpressing (MCF7/RAGE and T47D/RAGE) cells. Side panels show densitometric analysis of the blots normalized to β-actin, which was used as a loading control. D-E Evaluation of EphA3 protein expression (red signal) by immunofluorescence experiment in wild type (MCF7/wt and T47D/wt) and RAGE-overexpressing (MCF7/RAGE and T47D/RAGE) cells; nuclei were stained by DAPI (blue signal). The images shown represent 10 random fields from three independent experiments. Bottom panels represent corrected total cell fluorescence (CTCF), which was calculated on at least 10 pictures from each sample. Scale bar: 25 μm. F Schematic representation of human EPHA3 promoter carrying the Sp1-responsive sites (the transcriptional start site is indicated as + 1). G-H Protein expression of Sp1 in wild type (MCF7/wt and T47D/wt) and RAGE-overexpressing (MCF7/RAGE and T47D/RAGE) cells, as evaluated by immunoblotting. I-J Protein levels of EphA3 in the presence of 100 nM Sp1 inhibitor mithramycin A (MMA) in RAGE-overexpressing (MCF7/RAGE and T47D/RAGE) cells. Side panel shows densitometric analysis of the blots normalized to β-actin. K Recruitment of Sp1 to EphA3 promoter by ChIP assay in wild type (MCF7/wt and T47D/wt) and RAGE-overexpressing (MCF7/RAGE and T47D/RAGE) cells in the presence or absence of 100 nM Sp1 inhibitor mithramycin A (MMA). In control samples nonspecific IgGs were used instead of the primary antibody. The amplified sequences were evaluated by real-time PCR. Values represent the mean ± SD of three independent experiments performed in triplicate. (*) indicates p < 0.05