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. 2013 May 26;2013:161628. doi: 10.1155/2013/161628

Figure 1.

Figure 1

Ovatodiolide inhibits β-catenin signaling. (a) The structural formula of ovatodiolide. (b) TOP-flash, NF-AT, CRE, and NFκB reporter assays to evaluate the specificity of ovatodiolide. After 24 hr of transfection with a reporter plasmid, cells were exposed for 24 hr to DMSO or 20 μM ovatodiolide with agonist (LiCl (20 mM) for Top-flash, ionomycin (1 μg/mL) for NF-AT luc, forskolin (10 μM) for CRE luc, and TNFα (10 ng/mL) for NFκB luc activity controls) or vehicle control. Luciferase activity was assayed at 48 hr after transfection. (c) Western blot assay of effect of 40 μM ovatodiolide on protein levels of active β-catenin (p-β-catenin [S552]) and its downstream genes (c-myc, cyclin D1, and survivin) and other WNT molecules (TCF4, LRP5/6, p-LRP5/6, Axin1, and dishevelled) in RCC cell lines 786-O and Caki-1. (d) Quantitative real-time PCR of mRNA levels of β-catenin signaling target genes Axin2, Sp5, and Nkd1 in RCC cells after 48 hr of ovatodiolide treatment. All experiments were performed in triplicate. Data are mean ± SD from triplicate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.