Bili Inhibits Wnt Signaling Figure 3. Bili’s effect on Wnt signaling is conserved in zebrafish. (A) Wnt8 overexpression phenotypes with increasing severity. Embryos were scored as wt (green), small eyes (yellow), no eyes (orange) or severe (red). (B) Morpholino effect on in vitro expression of human Bili (hBili) or zfBili protein (control (como) or zfBili morpholino (bilimo)). (C) In the top panel, the effect of bilimo (loss of function) on Wnt8 GOF phenotype compared to como, is shown (n = 73 low Wnt dose, n = 147 medium Wnt dose, n = 57 high Wnt dose). Bili GOF (bili rna) rescues Wnt8 overexpression phenotype (control Renilla (co rna)). (n = 125 per dose of Wnt8) (bottom panel). doi:10.1371/journal.pone.0006129.g003 luciferase reporter and Renilla luciferase normalization control. Activation of the reporter in RKO cells following treatment with WNT3A conditioned media was enhanced by transfection of either hBili or AXIN1 siRNA and repressed by b-catenin siRNA (Fig. 4A). Similarly, activation of the reporter in HEK293T cells by transfection of WNT1, a pathway activator, was enhanced by hBili siRNA and repressed by b-catenin siRNA (Fig. S5A). Consistent with these loss-of-function studies, overexpression of hBili in HEK293T cells inhibited WNT3A-mediated activation of the reporter (Fig. 4B). A series of additional controls confirm these results and the conclusion that Bili is a negative regulator of the Wnt/b-catenin pathway in human cells. First, hBili siRNA effectively reduced both hBili mRNA and tagged protein levels. By qRT-PCR, hBili siRNA depleted hBili transcript levels by 50% (Fig. S5B), and reduced levels of overexpressed hBili protein (Fig. S5C). Second, four additional independent siRNAs designed against hBili had a similar or greater effect on Wnt-mediated activation of the b- catenin responsive reporter (Fig. S5D and E and data not shown) ruling out the formal possibility that the activity of the first siRNA was due to off-target effects. Third, hBili siRNA synergized with WNT3A conditioned media to induce transcriptional upregulation of the endogenous b-catenin target gene, AXIN2 (Fig. S5F). This effect on expression of an endogenous b-catenin target gene validates the conclusions of the synthetic reporters. Fourth, knockdown or overexpression of hBili had no effect on control b-catenin unresponsive reporters (Fig. S6A andB) or a CREB reporter that was activated with forskolin (Fig. S6C). These data collectively support the conclusion that Bili is a conserved inhibitor of Wnt/b-catenin signaling in diverse species. Bili functions upstream of b-catenin stabilization and associates with LRP6 We next carried out epistasis experiments in HEK293T cells expressing a b-catenin responsive luciferase reporter and Renilla luciferase normalization control. hBili or GFP were overexpressed PLoS ONE | www.plosone.org 5 July 2009 | Volume 4 | Issue 7 | e6129