Fig. 1

A) Scheme for high throughput screen. The primary assay quantified inhibition of Wnt3a in an RKO cell line engineered with a TCF-responsive firefly luciferase reporter and internal control Renilla luciferase. 76,000 small molecules were screened (see Methods). To rule out non-specific inhibitors, a NF-κB responsive luciferase reporter assay was used as a counter screen (see Methods). Inhibitors likely to act at or downstream of β-catenin stabilization were selected based upon inhibition of the GSK3 inhibitor BIO in the RKO TCF-luciferase primary screen assay.

B) Structures of PAWI-1, PAWI-2 and an IWR-1 analogue (Lanier et al., 2012; Willems et al., 2011). IC₅₀ values for inhibition of Wnt3a (100 ng/ml)-stimulated Super TOPflash TCF-luciferase reporter activity were determined in HEK293T cells; n=6; error bars, s.e.m.

C) PAWI-1 (500 nM) effect on induction of Wnt/β-catenin target genes c-MYC, CCND and AXIN2, as compared to DMSO vehicle alone control, in SW480 cells that have endogenous Wnt signaling. n=4; error bars, s.e.m. * indicates p-value <0.05 (T-test).

D) Effect of PAWI-1 (500 nM) on NF-κB, Notch and TGFβ signaling pathways (black bars), as determined using specific luciferase reporters in HEK293T cells (see Methods) and compared to DMSO vehicle controls (light bars). n=4; error bars, s.e.m. * indicates p-value <0.05 (T-test).

E) Effect of PAWI-1 (500 nM) on adult zebrafish tail fin regeneration. Compound was present for first 7 days post amputation (dpa) and removed for remaining time to 30 dpa (washout). PAWI-1 inhibited regeneration at 7dpa, but regeneration resumed after washout (16 and 30 dpa) indicating reversibility. Arrowheads (red) indicate the point of resection.