Fig 2: RAI2 suppressed activation of Wnt/β-catenin signaling pathway. (A) Dual-Luciferase reporter assay analyzed the effect of RAI2 plasmid transfection on wild-type β-catenin (β-catenin-wt)-induced activation of Wnt/β-catenin signaling in LoVo/HCT116 cells with or without LiCl/XAV939 addition (the concentrations of LiCl and XAV939 were 20mM and 10 µM respectively). LiCl and XAV939 were agonist and antagonist of Wnt/β-catenin signaling respectively. (B) Western blot analysis of RAI2, β-catenin, phosphorylated β-catenin (p-β-catenin) CtBP2 and the Wnt/β-catenin signaling target genes c-Myc, cylinD1, ASCL2, and LGR5 expression in LoVo/HCT116 cells with RAI2/empty vector transfection and RAI2-transfected cells with LiCl addition (20mM). The same experiment was also performed in shNC/shRAI2-transfected SW620 cells and shRAI2-transfected SW620 cells with XAV939 addition (10 µM). p-values: **<0.01; ***<0.001.

Fig 3: Mechanisms model for the regulation of RAI2 in Wnt/β-catenin signaling. ① RAI2 inhibits the interaction of CtBP2 and APC. ② RAI2 suppresses the expression of CtBP2.

Fig 4: RAI2 expression is negatively correlated with that of CtBP2. (A) Upper panel: A schematic of RAI2 domain organization and the mutant sites of mutant type RAI2 (RAI2-M); lower panel: Coimmunoprecipitation (Co-IP) analysis of RAI2 and CtBP2 interaction by anti-RAI2 antibody using cell lysates from LoVo cells transfected with empty vector, RAI2 expression vector or RAI2 mutant vector. And Cell lysates from SW620 cells were also used for Co-IP detection to analyze the endogenous interaction between CtBP2 and RAI2 by RAI2 antibody, with IgG group as a negative control. Input: before any antibodies were added. (B) The reciprocal mRNA expression between CtBP2 and RAI2 in 434 cases of colorectal cancer tissues from the TCGA dataset. (C) RT-PCR detection of RAI2 and CtBP2 expression in LoVo/HCT116 cells with or without RAI2 re-expressed and in SW620 cells with or without RAI2 knocked down.

Fig 5: Low expression of RAI2 was associated with a low level of phosphorylation of β-catenin and poor outcome in CRC. (A) Left panel: IHC staining of RAI2, phosphorylated β-catenin (p-β-catenin), ASCL2, and LGR5 in CRC tissues; right panel: the correlation between RAI2 expression and expression of p-β-catenin, ASCL2, and LGR5 (Spearman rank correlation test). (B) Kaplan-Meier curves show the association of the 5-year overall survival (OS) rate and relapse-free survival (RFS) rate of colorectal cancer patients with the expression status of RAI2. Black, RAI2-low expressed colorectal cancer patients (n = 101, log-rank test); red, RAI2-high expressed colorectal cancer patients (n = 197, log-rank test).