Figure 4.

FMOD promoter contains specific sites for binding by β-catenin/TCF4 to activate FMOD transcription which is promoted by the Wnt/β-catenin pathway and inhibited by Aspirin. (A) Nucleotide sequence of FMOD promoter sequence gi|568815597:c203340720-203339621 Homo sapiens chromosome 1, GRCh38.p7 Primary Assembly. Three putative β-catenin/TCF4 binding sites are indicated (boxed). (B) Quantification of FMOD promoter activity. MDA-MB-231 cells were cotransfected with FMOD-LUC reporter plasmid for 12 hours, and then were treated with or without 10 mM LiCl for 6 hours before treatment with DMSO or 5 mM Aspirin for 24 h. Luciferase activity was measured (see Materials and Methods) and normalized to GAPDH. (C) Quantification of FMOD promoter activity in HEK 293T cells that had TCF4 binding site core sequence deleted individually or in combination. HEK 293T cells were treated with LiCl in combination, with wild-type or mutant FMOD-PGL3 plasmids. After 24 hours the luciferase activity was measured and normalized to GAPDH. (D and E) CHIP assay (see Materials and Methods) was performed to verify the physical association of endogenous β-catenin and TCF4 with FMOD promoter sequence in MDA-MB-231 cells. ChIPs from fragmented chromatins of MDA-MB-231 cells were incubated with IgG, β-catenin and TCF4 primary antibodies. The purified DNA was analyzed by qPCR using the indicated primers (see SI). Data is shown as the mean ± SD of three to eight independent experiments. Student two-tailed t-test was used for statistical analysis (**P < 0.01, and ***P < 0.001).