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. 2021 Sep 10;11:716055. doi: 10.3389/fonc.2021.716055

Figure 4.

Figure 4

Gal-1 promotes β-catenin expression, nuclear translocation, and activity in CRC cells. (A) IF staining for β-catenin (green fluorescence) in KM12C with the addition of increasing doses of rhGal-1 for 48 hours. Nuclei were stained with Hoechst 33342 (blue fluorescence). Arrows show nuclear β-catenin; scale bar, 10 μm. (B) Western blot for β-catenin levels in whole cell lysate (top panel) and nuclear fraction (bottom panel) of KM12C with the addition of increasing doses of rhGal-1 for 48 hours; for nuclear fraction, histone H1 is used as the positive control and α-Tubulin as the negative control. (C) Luciferase reporter assay for β-catenin activity in KM12C with the addition of increasing doses of rhGal-1. TOPFlash plasmids (β-catenin promoter reporter construct containing TCF/LEF binding sites; please see Materials and Methods) and TOPFlash mutant plasmids (β-catenin promoter reporter construct containing mutated TCF/LEF binding sites; please see Materials and Methods) were transduced into KM12C, with the luciferase activity measured 48 hours later; addition of the Wnt/β-catenin agonist CHIR-99021 (CHIR; 0.3 µM) was used as a positive control. (D) qPCR analysis for the gene expression of Twist1 in KM12C after treatment with rhGal-1 (100 ng/ml) and without or with the Wnt/β-catenin antagonist XAV-939 (XAV; 10 µM) for 24 hours. All results are shown as the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01, and ***p < 0.005 compared to the control.