Skip to main content
. 2020 Aug 17;123(10):1521–1534. doi: 10.1038/s41416-020-1022-4

Fig. 3. Activation of the PI3K-Akt-GSK-3β-β-catenin signalling cascade is essential for Galectin-3-induced angiogenesis and EMT of HCC cells.

Fig. 3

a IF staining shows the colocalisation of β-catenin (green) and Galectin-3 (red) in Hep3B-vector, Hep3B-Galectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. Scale bars, 20 μm. b Western blotting shows the expression of phospho-PI3K, PI3K, phospho-Akt, Akt, phospho-GSK-3β and GSK-3β in Hep3B-vector, Hep3B-Galectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. c, d Hep3B-Galectin-3 cells were treated with DMSO, PF294002 (20 μmol/L), AZD5363 (10 μmol/L) or AR-A014418 (20 μmol/L). Western blotting shows the expression of Galectin-3, phospho-PI3K, PI3K, phospho-Akt, Akt, phospho-GSK-3β and GSK-3β (c). Western blotting of the expression of β-catenin in the whole cell, cytoplasm and nucleus (d). e, f Statistical diagrams of the tube formation assay, Transwell migration assay and cell adhesion assay. g, h Proteome angiogenesis array shows angiogenesis-related soluble proteins (g), and PCR array and western blotting show EMT-related genes/proteins (h) in Hep3B-shControl and Hep3B-shβ-catenin cells based on Galectin-3 overexpression. i Analysis of the IGFBP3 and VIM promoter identified a TCF4-binding site. Moreover, CHIP was performed using IgG and TCF4 antibodies, followed by qPCR in Hep3B-vector, Hep3B-Galectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. j IF staining shows the colocalisation of β-catenin (green) and IGFBP3 (red) (left) or vimentin (red) (right) in Hep3B-vector, Hep3B-Galectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. Scale bars, 20 μm. k Western blotting shows the expression of β-catenin, IGFBP3 and vimentin in Hep3B-vector, Hep3B-Galectin-3, Hep3B-Galectin-3-shControl and Hep3B-Galectin-3-shβ-catenin cells (upper) and Huh7-shControl, Huh7-shGalectin-3, Huh7-shGalectin-3-vector and Huh7-shGalectin-3-β-catenin cells (bottom). Ctrl control, G3 Galectin-3. The results represent three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.