Fig. 3

LASS2 attenuates Wnt/β-catenin signaling by inhibiting β-catenin nuclear translocation. A Screening of differentially activated pathways in LASS2-overexpressing BCSCs versus control BCSCs by Cignal Finder 10-Pathway Reporter Array. B Heatmap showing the expression pattern of Wnt/β-catenin downstream genes in BCSCs transfected with the indicated plasmids. C, D Immunoblotting of the indicated cytoplasmic (C) or nuclear (D) proteins in the control or LASS2-overexpressing BCSCs treated with vehicle (PBS) or 250 ng/mL Wnt3a for 6 h. E Immunofluorescent staining images of the indicated cells treated with either Wnt3a (250 ng/mL) or vehicle (PBS) for 6 h. Scale bar, 20 μm. F, G Luciferase activity of the ABCC2-reporter plasmid in BCSCs transfected with the indicated plasmids and treated with or without Wnt3a (250 ng/mL) for 30 min. Data are presented as the means ± SD of three technical replicates. ***p < 0.001, determined by one-way ANOVA. H TOPFlash/FOPFlash reporter gene assay in control and LASS2-overexpressing BCSCs treated with Wnt3a (250 ng/mL) or vehicle (PBS) for the indicated periods. Data are presented as the means ± SD of three technical replicates. ***p < 0.001 versus vehicle, ns, not significant versus vehicle; #p < 0.05, ###p < 0.001, NS, not significant, determined by one-way ANOVA. I, J Upper panel: schematic illustrations of the primer fragments of the ABCC2 (I) and CD44 (J) promoters. Lower panel: control and LASS2-overexpressing BCSCs treated with or without Wnt3a (250 ng/ml) for 30 min were subjected to chromatin immunoprecipitation using antibodies against β-catenin and TCF4, followed by RT–PCR for ABCC2 (I) and CD44 (J) gene fragments. Data are presented as the percentage of input. Data are presented as the means ± SD of three technical replicates. ns, not significant, **p < 0.01, ***p < 0.001, determined by one-way ANOVA. K A diagram illustrating the possible mechanisms of LASS2 in regulating Wnt/β-catenin signaling