FOXD and FOXI proteins are Wnt pathway regulators.A, TOPflash assay in MCF7, SAOS2, and mesenchymal stem cells (MSCs). MCF7 and MSC were pretreated with 5 μM GSK3 inhibitor CHIR99021. SAOS2 were pretreated with Wnt3a and R-spondin 3 conditioned media. B, TCF/LEF target gene expression in HCT116 following FOXD1 or FOXI1 expression, as determined by qPCR. C, TOPflash assay in 293T following FOXD1 or FOXI1 depletion with two independent siRNAs. D, TCF/LEF target gene expression in 293T following FOXD1 depletion, as determined by qPCR. E, representative image of colony formation assays in HCT116 following FOXD1 or FOXI1 gain-of-function. F, quantification of colony numbers from n = 4 biological replicates. Data in all panels were analyzed using Dunnett’s post hoc test against EV or siControl following one-way ANOVA (∗∗∗p < 0.001, ∗∗ p < 0.01, ∗p < 0.05). LEF, lymphoid enhancer-binding factor; RLA, relative luciferase activity; TCF, transcription factor 7. See also Fig. S6.