Regulation of the DCBLD2/ESDN promoter by TFAP2A. (A) The genomic organization of the human DCBLD2/ESDN promoter (-2185/+89) is shown and the three highest score TFAP2A binding sites identified are framed and named 1 or 2 or 3. The Transcription Start Site (TSS) is considered +1. (B)(C) HeLa (B) and MDA-MB-231 (C) cells were transiently co-transfected with either pGL3-Basic (basic) or pGL3-ESDN-WT (ESDNWT) or pGL3-ESDN-del3 (del3) vectors together with pSP(RSV)TFAP2A (TFAP2A) or TFAP2AshRNA2 (shTFAP2A) or their respective control empty vectors (EV or shEV) to obtain TFAP2A over-expression or down-modulation. pRLTK (Renilla luciferase) vector was transfected along to evaluate transfection efficiency and perform normalizations. Forty-eight hours later Firefly Luciferase activity was measured and normalized against Renilla Luciferase. Fold Changes were calculated referring to the pGL3-basic control vector and expressed as Relative Luciferase Units (RLUs). 250 ng of TFAP2A or shTFAP2A or EV or shEV were transfected unless specified differently. Three independent experiments were performed in triplicate and a representative one is shown here. The error bars indicate the Standard Errors (SE) of the triplicates. A student's t test was performed to evaluate if the experiments were statistically significant. * pv < 0.05; ** pv < 0.01; *** pv < 0.001. TFAP2A levels were measured by Western Blot (WB) analysis. Glyceraldheyde-3-phosphate-dehydrogenase (GAPDH) expression was used for protein loading controls.