Fig 2: APC knockout in CRC cells impairs Wnt-induced ß-catenin destruction complex localization. (a) APC staining in RKO and RKO-APCKO cells. (b–d) Representative images of RKO-APCKO cells treated with UB or WB. Representative images and scoring shown for (b,e) ß-catenin, (c,f) Axin1, and (d,g) GSK-3ß. Data averaged from three independent experiments (n = 25 cells per condition). Error bars, SEM; Statistical analysis by t-test: *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar, 10 µm.

Fig 3: miR-128-3p targets Axin1, SFRP2, WIF1, SMURF2 and PP1c in NSCLC cells.(a) Targetscan tool showing schematic representation of putative binding sites for miR-128-3p in 3'-UTRs of Axin1, SFRP2, WIF1, SMURFS and PP1c. (b) WB analysis of the protein levels of Axin1, SFRP2, WIF1, SMURF2 and PP1c in the indicated cells. (c) By immunoprecipitation against Ago1, RIP analysis reveals the interaction of miR-128-3p with the 3'-UTRs of Axin1, SFRP2, WIF1, SMURF2 or PP1c mRNA to form miRNP complexes. IgG immunoprecipitation, as well as the interaction of miR-128-3p with GAPDH and 5s rRNA, were used as negative controls. (d) Luciferase assay of pGL3-Axin1-3'-UTR, pGL3-SFRP2-3'-UTR, pGL3-SMURF2-3'-UTR, pGL3-PP1c-3'-UTR or pGL3-WIF1-3'-UTR reporters in the indicated cells, co-transfected with increasing amounts (20 and 50 nM) of the indicated oligonucleotides. The sequence of the miR-128-3p mutant is shown. (e) Effects of restored expression of Axin1, SFRP2, WIF1, SMURFS and PP1c in miR-128-3p-overexpressing cells on luciferase activities of the TOP/FOP reporter and TGF-ß reporter. (f) Effects of restored expression of miR-128-3p target genes on cell migration and self-renewal measured by Transwell migration assay and sphere formation assay, respectively, in the indicated NSCLC cells. Error bars represent mean±s.d. derived from three independent experiments. A two-tailed Student’s t-test was used for statistical analysis (*P<0.05, **P<0.01).

Fig 4: Model of NAMPT’s role in the proliferation of human CRC cells through the regulation of Wnt/ß-catenin signaling. A high level of NAMPT (left panel) yields more NAD+ and induces downregulation of Axin and the nuclear translocation of ß-catenin, thereby leading to the activation of Wnt/ß-catenin signaling and the increased growth of CRC cells. However, when NAMPT is inhibited by genetic or pharmacological methods (right panel), the NAD+ level is reduced and Axin is upregulated. Then, ß-catenin is degraded, and the Wnt/ß-catenin pathway is suppressed, resulting in the inhibition of cancer cell proliferation

Fig 5: Inhibition of Wnt/ß-catenin reduces tumor malignant behaviors caused by DJ-1. (A) The proliferation of LV-DJ-1 cells treated with XAV939 was significantly decreased compared with LV-DJ-1 cells (magnification, x100). (B) In the colony formation assay, cells treated with XAV939 had fewer colonies compared with the control LV-DJ-1 cells (magnification, x100). (C) In the Transwell assay, cells of the LV-DJ-1 + XAV939 group demonstrated a weaker ability to migrate and invade compared with the LV-DJ-1 group (magnification, x200). (D) The adhesion ability was decreased in the LV-DJ-1 + XAV939 group compared with the LV-DJ-1 group. (E) Western blotting results demonstrated XAV939 treatment could increase the E-cadherin expression, while it reduced the vimentin and N-cadherin expression levels. (F) LRP6, p-LRP6 and ß-catenin expression were downregulated, while Axin1 expression was upregulated in XAV939-treated compared with control cells. The ratio of p-LRP6 to total LRP6 protein was significantly increased in XAV939-treated cells compared with the control untreated LV-DJ-1 cells. *P<0.05, **P<0.01 vs. LV-DJ-1 group. LV-DJ-1, lentivirus overexpressing DJ-1; LRP-6, lipoprotein receptor-related protein 6; p-. phosphorylated.