Gal-3 knockdown sensitizes cells to cell death in oxygen and nutrient deprivation in the NG97ht cell line.

<p>Analysis of NG97ht cells incubated with specific gal-3 siRNA or scramble siRNA for 6 h and then exposed to either normoxia (N), CoCl₂ (C) or hypoxia (H) in complete or serum-deprived medium for additional 42 h. <b><i>A</i></b>. Protein analysis demonstrated gal-3 knockdown in each experimental condition. Gal-3 knockdown was calculated relatively to its β-actin control. <b><i>B</i></b>. In addition, gal-3 mRNA was evaluated and qRT-PCR demonstrated significant knockdown upon treatment with specific siRNA compared to scramble siRNA. <b><i>C</i></b>. Analysis of hypodiploid cells demonstrated no differences in cell death rates in cells exposed to high serum concentrations, regardless the levels of gal-3 expression. <b><i>D</i></b>. Upon serum-deprivation, cells were sensitized to cell death in hypoxic conditions. Sensitization was much more pronounced upon knockdown of gal-3, as compared to cells transfected with scramble siRNA. T98G cells transduced with gal-3 shRNA demonstrated decreased gal-3 protein by western blot (<b><i>E</i></b>) and mRNA by qRT-PCR (<b><i>F</i></b>) compared to scramble shRNA. Gal-3 knockdown was calculated relatively to its β-actin control. <b><i>F</i></b>. Analysis of hypodiploid cells demonstrated increased cell death in cells transduced with gal-3 shRNA compared to cells transduced with scramble shRNA when exposed both to hypoxia or hypoxia and serum-deprivation. Representative experiment performed at least in three independent assays and data are presented as mean±SEM. **p<0.01, ***p<0.001.