Figure 6
Point mutations in the EGR1 and RXRA binding sites in the CCL2 promoter completely abolished its activation by TGF-β1. (A) Position weight matrixes of the EGR1 and RXRA binding sites and the strategy of their point mutagenesis. Red arrows indicate nucleotides changes. (B) Point mutations of the EGR1/RXRA1 sites in the CCL2 promoter totally abrogated EGR1 and RXRA1 binding to the promoter. The efficiency of EGR1 and RXRA1 binding to different variants of the CCL2 promoter was estimated by pull-down assay with the use of nuclear extracts from MDA-MB-231 and HCC1937 breast cancer cells. The data were normalized to the normal − 244/− 1 amplicon after subtraction of the background signal (the signal for this sample is represented as 1 for each cell line). The results of five independent experiments are represented. *P < 0.01. (C) Mutation of the EGR1/RXRA binding sites resulted in a full absence of the effect of TGF-β1 (10 ng/mL, 24 h) on CCL2 promoter activity in both the MDA-MB-231 and HCC1937 cell lines as estimated using a luciferase reporter assay. The data were obtained in five independent experiments and normalized to the Renilla luciferase activity. Normalized luciferase signals of original CCL2 promoter without TGF-β1 activation are represented as 1 for each cell line. *P < 0.01.
