Fig 2: CDK3 inhibits Wnt/β-catenin signaling in breast cancer cells(A) Gene ontology analysis of top 37 genes that had 2-fold decrease in MDA-MB-231 cells overexpressed CDK3, top 50 genes had 2-fold decrease in BT549 cells overexpressed CDK3, and top 50 genes had 2-fold increase in MCF7 cells silenced CDK3. (B) Western blot was performed to detect molecules expression involved in Wnt/β-catenin signaling in both CDK3 overexpressed MDA-MB-231 cells and CDK3 silenced MCF7 cells. (C) Transwell assay was performed in Wnt3a conditional medium or control medium treated MDA-MB-231 cells, and quantified data was shown below. (D) Western blot was performed to detect Wnt/β-catenin signaling in Wnt3a treatment (***P < 0.001, NS indicates not significant).

Fig 3: Suppression of CFTR activates β-catenin signaling pathway by interacting with DVL-2 in renal epithelial cells. (a) Western blot showing the increased nuclear accumulation of β-catenin in ribozymes-mediated CFTR knockdown HK-2 cells (Rib) comparing to control cells (His) transfected with control vectors (pEF6/V5-His); (N.E-nuclear extraction; C.E-cytoplasmic extraction. (Full-length blot is shown in Supplementary Figure S7g.) (b) luciferase activity assay showing knockdown of CFTR increases β-catenin transcriptional activity whereas overexpression of CFTR (pcDNA3.1 CFTRov) decreases β-catenin activity in HK-2 cells. Cells were lysed at 48 hours after transfected with TOPflash reporter gene (0.5 μg/ml) and Renilla-Luc (0.05 μg/ml) and the relative luciferase activity was defined as the ratio of readout for firefly luciferase to that for renilla luciferase with that of control group set as 1.0. *p < 0.05; (c) Real time-PCR showing increased mRNA expression of β-catenin target genes in CFTR knockdown HK-2 cells, **p < 0.01; (d) Western analysis showing increased protein expression of β-catenin target genes in the CFTR knockdown HK-2 cells. CFTR knockdown HK-2 cells (Rib) comparing to control cells (His) transfected with control vectors (pEF6/V5-His); (Full-length blot is shown in Supplementary Figure S7h.) (e) Western blot analysis shows that iCRT14 reverses decreased expression of E-cadherin in a dose-dependent manner in CFTR knockdown HK-2 cells; (Full-length blot is shown in Supplementary Figure S7i.) (f) Immunofluorescent staining showing the co-localization of CFTR and Dvl2 in HK-2 cells; (g) Co-immunoprecipitation showing the interaction of CFTR and Dvl2 in HK2 cells; (Full-length blot is shown in Supplementary Figure S7j.) (h) CO-IP of exogenous CFTR and Dvl2 in HEK293 cells showing lack of interaction when CFTR PDZ-binding domain is deleted; (TL: total cell lysates; full-length blot is shown in Supplementary Figure S7k.) (i) luciferase activity assay shows that overexpression of CFTR wild type (peGFP-CFTR), but not CFTR with deleted PDZ-binding domain (peGFP-CFTR delPDZ), increases β-catenin transcriptional activity in HK-2 cells. Cells were lysed at 48 hours after transfected with TOPflash reporter gene (0.5 μg/ml) and Renilla-Luc (0.05 μg/ml) and the relative luciferase activity was defined as the ratio of readout for firefly luciferase to that for renilla luciferase with that of control group set as 1.0. *p < 0.05.

Fig 4: Activation of the WNT pathway after BORIS over-expression. (A) Western blot analysis of WNT5A/B and TCF3 in untransfected control, mock transfected (C3-empty) or BORIS transfected (C3-BORIS) HEK293T cells. The upper band in the WNT5A/B blot is non-specific (*) as shown by provider (Cell Signalling). The membranes were probed with the antibodies as indicated and GAPDH was used as a loading control. Lysates from three independent experiments were loaded on the same gel. (B) mRNA levels of BORIS bound transcripts in HEK293T cells over-expressing BORIS (C3-BORIS) as compared to empty vector (C3-empty). Data in (B) represent 2 technical replicates ± SD. (C) Luciferase activity of a TCF/LEF-responsive element reporter after BORIS (C3-BORIS) or mock transfection of HEK293T cells. Over-expression of BORIS (C3-BORIS) led to a more than four-fold increase in luciferase activity compared to cells transfected with empty vector alone (C3-empty). Cignal negative is a non-inducible reporter, lacking the TCF/LEF response element. LiCl was used as a general activator of the TCF/LEF reporter. Data are expressed as a mean ratio of the Firefly/Renilla luciferase activity (see Materials and Methods) and represents the average ± SD of 4 biological replicates. (D) Western blot analysis of HEK293T cells after over-expression of BORIS together with siRNA knock down of β-catenin. LiCl treated cells (C3-empty + LiCl) were included in the analysis to exclude a direct effect of LiCl on the expression of β-catenin. GAPDH was used as a loading control. (E) Densitometry quantification of β-catenin expression in (D). Data represents the mean ± SD of 3 biological replicates. (F) Normalised TCF/LEF luciferase reporter activity after β-catenin knock-down and BORIS over-expression in HEK293T cells. β-catenin knock-down efficiently prevented BORIS-mediated activation of the TCF/LEF luciferase reporter, compared to Scrambled + Reporter with β-catenin siRNA + Reporter. Data shown is from one representative experiment.