Fig. 4

LASS2 suppresses the dephosphorylation of β-catenin by inhibiting PP2A activity and dissociating PP2A from β-catenin. A LASS2-overexpressing or control BCSCs were transfected with the indicated siRNAs, followed by treatment with MG-132 (10 μM) for 6 h. Cell lysates were harvested for immunoblotting assays. B LASS2-overexpressing or control BCSCs were treated with 20 μg/mL cycloheximide (CHX) for the indicated periods, followed by immunoblotting. The band density was normalized to the 0-time point. C Quantitative analysis of ceramides by LC–MS in BCSCs transfected with LASS2-overexpressing or control plasmids. D PP2A was activated in LASS2-overexpressing BCSCs by transfection with a PP2A-C subunit plasmid, and treatment with C18-ceramide (100 μM) or DT-061(10 μM) for 6 h. Cell lysates were harvested for immunoblotting assays. E–G LASS2-overexpressing or control BCSCs were treated with 100 μM C18- or C24-ceramide for 6 h. Cell lysates were harvested for immunoblotting assays (E, F) and PP2A immunoprecipitation phosphatase assays (G). H BCSCs were pretreated with MG-132 (10 μM) for 6 h, followed by treatment with Wnt3a (250 ng/mL), tautomycetin (1 nM), okadaic acid (0.1 nM), or NSC117079 (50 μM) for additional 2 h. PBS was used as a control. Cell lysates were harvested for immunoblotting assays. I Coimmunoprecipitation of β-catenin and the PP2A-B subunit in BCSCs with or without LASS2 overexpression. Data are presented as the mean ± SD of three independent experiments. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, determined by Student’s t test or one-way ANOVA