Figure 1. Xenopus egg extract screen identifies pyrvinium as an inhibitor of Wnt signaling.

(a) Chemical structure of pyrvinium pamoate. (b) Pyrvinium decreases cytoplasmic and nuclear β-catenin levels. HEK 293 cells were treated for 16 h as indicated, and fractionated lysates were immunoblotted for β-catenin. Purity of nuclear and cytoplasmic preparations was assessed by immunoblotting for acetylated histone H3 and actin, respectively. (c) Pyrvinium increases cellular Axin levels. Lysates from HEK 293 cells treated for 16 h as indicated were immunoblotted for Axin and actin (loading control). (d) Pyrvinium blocks Wnt-mediated nuclear accumulation of β-catenin. IEC-6 cells treated as indicted were stained for β-catenin and DNA. (e) Pyrvinium inhibits TOPflash activation with an EC₅₀ of ~10 nM. HEK 293 STF (TOPflash) or constitutively expressing luciferase (CMV-Luc) reporter cells were treated as indicated. Graph represents mean ± s.e.m. of TOPflash signal normalized to cell number (performed in quadruplicate). RLU, relative light units. (f) Pyrvinium decreases levels of endogenous Wnt target transcripts. Data shown represent mean of four independent real-time PCR reactions, graphed as relative expression to unstimulated cells and normalized to β-actin. Error bars, RQ (relative quantification) values >95% confidence.