Figure 2

The glycome of activated hepatic stellate cells (HSCs) facilitated Gal-1 binding which induces HSC migration and activation. (A) Gal-1 induces the migration of LX-2 cells in a dose-dependent manner. The cell migratory ability was measured using a Boyden chamber assay. (B) Gal-1 induces HSC activation. LX-2 cells were starved for 24 h and then treated with different doses of the recombinant Gal-1 protein for 24 h. α-smooth muscle actin (α-SMA) expression was detected using an RT-qPCR. Relative expression levels were calculated by comparing the ΔCT values of Gal-1-treated cells to those of cells without treatment, and results are shown as folds of change. (C) Thiodigalactoside (TDG) inhibits Gal-1 binding with LX-2 cells. TDG (200 µM) was pre-incubated with Gal-1-488 (500 nM) for 30 min, and then LX-2 cells were incubated with the mixture for 30 min. The binding of LX-2 and Gal-1-488 was analyzed using flow cytometry. (D) TDG inhibits Gal-1-induced HSC migration. TDG (200 µM) was pre-incubated with Gal-1 (500 nM) for 30 min, and then LX-2 cells were treated with the mixture for 16 h. The cell migratory ability was measured using a Boyden chamber assay. (E,F) Knockdown of Gal-1 normalizes activated HSCs. LX-2 cells were infected with a lentivirus carrying Gal-1 and luciferase shRNAs (sh-B09, D09, and sh-Luc). Western blotting and an RT-qPCR were used to analyze Gal-1, α-SMA, fibroblast activation protein (FAP), and α-1 type I collagen (COL1A1) expression. Relative expression levels of individual genes were calculated by comparing the ΔCT values of sh-B09 cells to those of sh-Luc cells, and results are shown as folds of change. All of the experiments were performed in duplicate. Results are shown the mean ± SEM of three independent assays. *p < 0.05.