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. 2013 Dec 12;42(5):2988–2998. doi: 10.1093/nar/gkt1275

Figure 5.

Figure 5.

β-Catenin/TCF/Lef-1 binds to and activates the promoter of miR-183-96-182 cluster gene. (A) Schematic illustration of the promoter region of the miR-183-96-182 cluster gene showing the locations of the core promoter and putative TBEs. The first nucleotide of miR-96 was set as 1. (B) ChIP assay experiments were performed using a SimpleChIP® Enzymatic Chromatin IP Kit and a rabbit mAb against β-Catenin. Five binding sites for β-Catenin/TCF/Lef-1 complex were confirmed in AGS cells. An extra site downstream of the putative core promoter was confirmed in HeLa cells. (C) A renilla luciferase construct was generated by subcloning the upstream TBEs containing a DNA fragment into a luciferase vector. Cotransfection of a construct encoding β-Catenin together with the luciferase vector into AGS cells increased the renilla luciferase activity while cotransfection of a construct encoding GSK3β had the opposite effect (compared with EV, *P < 0.05 by Student’s t-test). (D) Knockdown of β-Catenin significantly decreased renilla luciferase activity, while knockdown of GSK3β increased renilla luciferase activity. The same luciferase construct as in Figure 4C was cotransfected with β-Catenin siRNA or GSK3β siRNA, respectively, into AGS cells (compared with control siRNA, *P < 0.05 by Student’s t-test). All experiments were repeated three times with similar results.