Fig 2: Basic characterization of PLC-PRF-5 cells with rs290487 C/C and rs290487C/T genotypes. (A) QRT-PCR analysis results show significant reduction in the expression of total TCF7L2 total mRNA and liver-specific alternative spliced transcripts containing exons 11-12-13, exons 11-13-14, and exon 8+15bp in the C/C cells compared to the C/T cells. (B) Dual luciferase reporter assay results show reduction in relative luciferase activity in cells transfected with pGL3-basic-promoter-rs290487 C/C transfected with pGL3-basic-promoter-rs290487 T/T. (C) Western blot results show that TCF7L2 protein levels are reduced in C/C cells compared to the C/T cells. (D) Western blot results show that TCF7L2 protein levels are increased in insulin- and high glucose- treated C/C and C/T cells. Note: * P< 0.05; ** P< 0.01.

Fig 3: The levels of FBXW2 and ß-catenin predict patient survival. a, b A general tendency of inversed protein levels between FBXW2 and ß-catenin in lung tumors: Human lung cancer tissue microarrays containing 90 human lung cancer specimens were immune-stained with anti-FBXW2 and anti-ß-catenin antibodies. Representative staining pictures of tumors are shown (a). Quantification of the staining intensity was performed by semi-quantitative scoring with all 90 tumors (r = –0.6861 p < 0.001, Pearson correlation coefficient) (b). c The expression levels of FBXW2 are reduced, while the expression levels of ß-catenin are increased in the tumors with lymph-node metastasis: The staining intensity of FBXW2 and ß-catenin in tumors with or without lymph-node metastasis (N = 0: without lymph-node metastasis, N ? 0: with lymph-node metastasis) were statistically analyzed. Data are shown as mean ± s.d. ***p < 0.001 (Student’s t-test). d The association of FBXW2 and ß-catenin expression with overall survival in lung cancer patients. A total of 180 lung cancer tissue microarray samples were stained for FBXW2 and ß-catenin levels. The Kaplan-Meier analysis was performed in 88 cancer samples showing an inverse correlation. The results showed that lower levels of FBXW2, coupled with higher levels of ß-catenin, predict a worse overall patient survival (p = 0.0243, log-rank test). e Working model. EGF activation of EGFR leads to activation of AKT1, which inactivates GSK-3ß to abrogate its binding with ß-TrCP, but phosphorylates ß-catenin at Ser552 to facilitate its binding with FBXW2 for subsequent ubiquitylation and degradation. In human cancer (e.g. lung cancer) with low level of FBXW2, phospho-ß-cateninser552 is not degraded and translocated, along with non-degraded wild type ß-catenin, to the nucleus, and forms a complex with TCF4M/S or TCF4E, leading to transactivation of the genes encoding MMPs or c-Myc/Cyclin D1 for enhanced migration/invasion and proliferation, respectively (see text for more details)

Fig 4: Suppression of the canonical WNT in the wild type and Myh6-Cre:DspW/F (cardiac myocyte-specific heterozygous deletion of the Dsp gene) mice. (A) Transcript levels of Axin2, a bona fide target of the cWNT pathway in the wild type and Myh6-Cre:DspW/F mice treated with placebo and WNT974 along with untreated controls, as determined by RT-PCR. Relative fold change in the transcript levels was determined by comparing all the groups to the wild type untreated group. (N = 5–7 per group). (B) Representative immunofluorescence images depicting expression and nuclear localization of TCF7L2 protein, a co-transcriptional regulator of the canonical WNT pathway, in the myocardial sections in the experimental groups. (C) Quantitative data showing the percentage of cells expressing TCF7L2 in the myocardial sections in the experimental groups. The P values were determined by the student t-test between two groups and genotype-by-treatment interactions were determined by 2-way ANOVA. The significant P values (< 0.05) between the groups are presented. PG: P value for the effect of the genotype; PTr: P value for the effect of treatment; Pi: P value for treatment-by-genotype interaction.

Fig 5: TCF7L2 directly regulates terminal effector genes in the thalamus. (A) Enrichment analysis of the TCF7L2 consensus motif in the TCF7L2 ChIP peaks by the AME algorithm. TP, true positive peaks, where the TCF7L2 motif was identified; FP, false positive peaks, where the TCF7L2 motif was identified in reshuffled peaks data. (B) De novo motif discovery analysis. The most significant motif identified by MEME-ChIP is TCF7L2. (C) Venn diagram showing the overlap between genes identified by RNA-seq (red) and ChIP-seq (blue). The P60 DEGs are in white, DEGs overlapping with ChIP-seq are in black. (D) Venn diagrams showing the overlap between the upregulated (red) or downregulated (green) P60 DEGs and genes identified by ChIP-seq (grey). (E) TCF7L2 binding profiles in the Cacna1g, Gabra4, Slc17a7 (Vglut1), Grin2b, Rora and Lef1 genes. Two independent biological replicates are represented as separate lines: pink/blue, the TCF7L2 ChIP-seq signal; orange/olive, input; green, negative control (IgG).