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. 2015 Oct 19;10(2):224–239. doi: 10.1016/j.molonc.2015.10.005

Figure 4.

Figure 4

β‐catenin directly regulates MUC4 transcription. (A) A stabilized β‐catenin construct (4ACAT; S33A, S37A, T41A, T45A) elicited increased TOP/FOP luciferase activity in comparison to the empty vector control in CD18/HPAF cells. (B) Luciferase activity for MUC4 promoter fragments p3778, p3000, and p2700 in the presence of 4ACAT compared to the empty vector control. Luciferase readings are the average of three or more separate experiments performed in triplicate; the standard deviation shown is for three or more separate experiments. (C) The p3778 promoter construct with each of the three putative TCF/LEF sites mutated (i.e., −2612:MUT1, −3226:MUT2, −3408: MUT3) was transfected into CD18/HPAF cells in the presence of 4ACAT. The pCMV9‐Renilla vector was used as an internal transfection control; all luciferase experiments were performed in triplicate and repeated a minimum of three times. Images represent the average of at least three experiments, each performed in triplicate. *p = 0.02, **p = 0.002 (D) Quantitative ChIP assay using β‐catenin antibody to pull down sheared chromatin isolated from CD18/HPAF Scr and sh‐cat. Primer pairs specific to the TCF/LEF Sites #1, #2, and #3 on the MUC4 promoter were used. Primers amplifying the TCF/LEF site on the c‐Myc promoter were used as a positive control; an unrelated primer pair was used as a negative control. All real‐time values were normalized to the 1% input control.