Fig 2: Inhibition of ALDH1A2 expression by GSK3 inhibitor CHIR99021 is via ß-catenin signaling. (A) Effect of CHIR99021 on ß-catenin nuclear translocation in WiT49 cells. WiT49 cells were treated with 5 µM CHIR99021 or 0.1% DMSO for 48 h, and then immunofluorescence was performed with ß-catenin antibody and DAPI staining. Negative control represents no primary antibody control. Bar length equals 50 µm. (B) Effect of CHIR99021 on the TCF reporter in WiT49 cells. A 500-ng sample of TOPFlash or FOPFlash constructs, together with 50 ng of the pRL-TK renilla control vector, was transiently transfected into WiT49 cells. Cells were treated with 0.1% DMSO or 5 µM CHIR99021 after 24 h of transfection. Luciferase reporter activities relative to renilla (pRL-TK) are shown in percentage terms. Values are the mean ± SD of three determinations from separate experiments. ***P < 0.001 compared with the DMSO control group (without CHIR99021 addition). (C,D) Effect of CTNNB1 knockdown on the downregulation of ALDH1A2 by CHIR99021 treatment. WiT49 was transfected with 50 nM CTNNB1 siRNA or negative control siRNA for 24 h, followed by treatment of 5 µM CHIR99021 for another 48 h. CTNNB1 and ALDH1A2 mRNA expression relative to TBP control in WiT49 cells were measured by real-time RT-PCR (C). Values are the mean ± SD of three determinations from separate experiments. ***P < 0.001 compared with the siNEG control group. ß-catenin (CTNNB1), ALDH1A2 and GAPDH protein expression in WiT49 cells were measured by Western blot (D). Values under bands were calculated by the density of bands of ß-catenin or ALDH1A2 divided by the density of bands of GAPDH.

Fig 3: Bcl-3 activates Wnt/ß-catenin signalling pathway and maintains ß-catenin functions in vitro. (A,B) Gene ontology (GO, Left) and Kyoto encyclopedia of genes and genomes (KEGG, Right) analysis of downregulated genes with Bcl-3 deletion in bone marrow mesenchymal stem cells (BMSCs). (C) Heat map showed relative genes expression in Wnt pathway of shCtrl and shBcl-3 BMSCs. (D) Gene set enrichment analysis (GSEA) showed a significant decrease of Wnt/ß-catenin gene signatures in shBcl-3 BMSCs. (E) Lysates from BMSCs were used, and anti-Bcl-3 antibody was used in IP followed by immunoblot using the indicated antibodies. (F) Bcl-3, Ac-K49-ß-catenin and ß-catenin were analysed by immunoblot in control and Bcl-3-knockdown BMSCs after treated with Wnt 3a for 0 h or 4 h. (G) The levels of Bcl-3 in nuclear and cytoplasmic. (H) Bcl-3, Ac-K49-ß-catenin and ß-catenin were analysed by immunoblot in control and Bcl-3-knockdown BMSCs after treated with cycloheximide (CHX, 50 mg/ml). (I) BMSCs were cotransfected with the indicated siRNA and TOP/FOP Flash reporter plasmid. (J) ChIP assays on the promoter regions of the Runx2 and Osterix genes were performed in control and Bcl-3-silenced BMSCs. The data are presented as the mean ± SD. **P < .01; ****P < .0001 vs. control group. Statistical analysis was performed using two-way ANOVA. Primary antibodies: GAPDH, Abcam(ab181602); Lamin B1, Abcam(ab133741); ß-catenin, Proteintech (51067-2-AP); K49 acetyl-ß-catenin, Cell Signalling Technology(9030); BCL-3, Abcam(ab259832)

Fig 4: SOST knockdown activates Wnt/ß-catenin signaling in UM cells. (A, B) Western blot of SOST expression in APRE-19 cells as well as three human UM cell lines OCM-1, Mum-2B, and Mum-2C. (C–H) RT-qPCR of the expressions of WNT1, CTNNB1, CCND1, MMP2, and MMP9 mRNAs in si-SOST-transfected OCM-1, Mum-2B, and Mum-2C cells and controls. (I-O) Western blot of SOST, Wnt-1, ß-catenin, Cyclin-D1, and MMP2 as well as MMP9 levels in si-SOST-transfected OCM-1, Mum-2B, and Mum-2C cells and controls. Ns, no significance; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig 5: SETD1A activates the Wnt/ß-catenin pathway by stabilizing ß-catenin. A-B, MYC, CCND1 and nuclear ß-catenin levels in NSCLC cells as indicated were analyzed by western blotting. C, CTNNB1 transcript levels in NSCLC cells as indicated was analyzed by qRT-PCR. ns, not significant. D, Subcellular localization of SETD1A and ß-catenin in NSCLC cells was analyzed by confocal laser scanning microscope. The subcellular distribution of SETD1A and ß-catenin was quantified by Image J software. E, The interaction between SETD1A and ß-catenin in PC9 cells was analyzed by CoIP assay. F-G, ß-catenin stability in the SETD1A knockdown and negative control group PC9 cells was analyzed by CHX chase assay. H, Ubiquitination of ß-catenin in PC9 cells was analyzed by CoIP assay following SETD1A knockdown. I, Two-step co-immunoprecipitation of the complex containing SETD1A, ß-catenin and PKA is shown. HEK293T cells were cotransfected with the Flag-tagged SETD1A plasmid and HA-tagged ß-catenin plasmid. The first immunoprecipitation was performed using anti-FLAG M2 beads. The complex was eluted using 3 × Flag peptide followed by the second step of co-immunoprecipitation with anti-HA antibody. Then the protein samples were analyzed by western blotting with anti-Flag, anti-HA and anti- anti-PKAa cat antibody. Data are shown as means ± SD.