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. 2021 Jun 15;13(12):2986. doi: 10.3390/cancers13122986

Figure 2.

Figure 2

N6L inhibits β-catenin activation. (A–C) mPDACs, MIA PaCa-2 and Panc-1 cells were transfected with Firely luciferase Wnt reporter and Renilla luciferase for control and stimulated with Wnt3a-CM in presence or absence of N6L for 24 h. Graphs show the fold change of the ratio of Firefly/Renilla luciferases activity (Dual-Luciferase Reporter Assay). Wnt3A-CM induced a significant increase of luciferase activity that was inhibited in presence of 30 µM N6L in all cell lines. (One-way ANOVA, multiple comparison, n = 3 experiments, **** p < 0.001). (D–G) Western blotting of active, total β-catenin and phospho-Ser9-GSK3β of MIA PaCa-2 cells treated for 3 h with N6L and Wnt3a-CM and relative quantification are shown. Active β-catenin was analyzed by using an antibody against non-phosphorylated form of β-catenin. In (E–G) (One way ANOVA, multiple comparison, n = 5, ns = non-significant, * p < 0.05, ** p < 0.01, **** p < 0.0001). (H,I) Western blotting and relative quantification of active β-catenin level of MIA PaCa-2 cells treated for 30 min with 10 µM TWS119 alone or with 30 µM N6L (One-way ANOVA, multiple comparison, n = 3, * p < 0.05, ** p < 0.01). (J) qPCR analysis of AXIN2 mRNA and (K,L) Cyclin D1 protein level analysis by Western blotting after 24 h of N6L treatment in presence of Wnt3A-CM compared to Wnt3a-CM alone. (One-way ANOVA, multiple comparison, n = 3, ns = non-significant, * p < 0.05, ** p < 0.01, *** p < 0.005).