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. 2024 Sep 4;15(9):654. doi: 10.1038/s41419-024-07028-3

Fig. 1. Knockdown of LGALS8 potentiates the migratory characteristics of TGF-β responsive HT29 cells by increasing RAS and Src activities.

Fig. 1

A Scoring results from IHC staining of galectin-8 in tumor tissues from CRC patients classified by T-classification. B Western blotting shows the expression of galectin-8 protein in various CRC cells, FHC and normal human intestinal epithelial cells (HcoEpi). Cell adhesion activities measured 6 h after seeding (C) and migration activities measured 24 h after seeding (D) of HT29 cells expressing shCtrl or shGal-8 (top) or transfected with a mixture of three different siRNAs targeting galectin-8 (siGal-8) or a scrambled control siRNA (siCtrl) (bottom) were analyzed by xCELLigence RTCA. E Western blotting shows the expression of EMT markers in the whole cell lysate and nuclear lysate isolated from the mock-treated, shCtrl-expressing, and shGal-8-expressing HT29 cells. Actin and Lamin A serves as the whole cell lysate and nuclear lysate loading control, respectively. α-tubulin serves as the negative control of nuclear lysate preparation. Cell migration activities of shCtrl- and shGal-8-expressing HT29 cells (F) or siCtrl- and siGal-8-transfected HT29 cells (G) in the presence or absence of TGF-β (10 ng/mL), the pan-RAS inhibitor lonafarnib (RAS-I, 100 μM), and the Src inhibitor dasatinib (SRC-I, 30 μM), assessed by xCELLigence RTCA at 48 h after seeding. H Proximity ligation assay (PLA) to determine the interaction of endogenous galectin-8 with TβRII in HT29 cells. Scale bar = 10 μm. I HT29 cells were treated with or without DTSSP crosslinker before cell lysis. Co-immunoprecipitation (Co-IP) was used to examine the interaction of galectin-8 and TβRII by IP of lysates with anti-TβRII antibody or isotype control mouse IgG, followed by immunoblotting (IB) with the indicated antibodies. J Western blotting shows the expression of activated, phosphorylated, and total proteins of RAS or Src in mock-treated, shCtrl, shGal-8 alone or with LY2109761 treated HT29 cells. Actin serves as the loading control. Migration activities of HT29 cells expressing the indicated shRNA (K) or siCtrl- and siGal-8-transfected HT29 cells (L) and/or treated with TGF-β signaling antagonist LY2109761 (10 µM), RAS-I (100 μM), and SRC-I (30 μM) for 48 h. Results in C, D, F, G, K, L are shown as mean ± SD (3 biological replicates with technical duplicates in C (left), D (top), G and K; 3 biological replicates with technical tripilicates in C (right) and D (bottom); 3 biological replicates with technical quintuplicates in F; 2 biological replicates with technical duplicates in L). Statistical tests were calculated by one-way ANOVA for A and t-test for C, D, F, G, K, L. ns not significant.