FIG. 8.

Endogenous galectin-3 regulates FN fibrillogenesis and the conformation and association of α5β1-integrin with fibrillar adhesions. (A) Expression of endogenous galectin-3 in Mgat5^+/+, Mgat5^−/−, and rescued cells was assessed by Western blotting relative to β-actin expression levels. Molecular mass markers (in kDa) are indicated, and the graph shows the densitometric quantification of band intensity (±SEM; n = 4; P values are relative to Mgat5^+/+ cells). Mgat5^+/+ cells were plated for 2 days on glass coverslips coated with 10 μg/ml of FN in serum-free medium (CTL), in serum-free medium supplemented with anti-galectin-3 monoclonal supernatant (1:50 dilution; +α-Gal-3), or in the presence of anti-galectin-3 antibody plus 20 mM lactose (+α-Gal-3+β-lac) or 20 mM sucrose (+α-Gal-3+Suc). Cells were then labeled for β1-integrin (green) and phalloidin (red) (B), SNAKA51 (green) and FAK (red) (C), or FN (green) and phalloidin (red) (D). Corresponding graphs show the number of cells presenting β1-integrin-labeled fibrillar adhesions (B), SNAKA51 labeling intensity (C), and FN fibril expression (D) in control and treated cells (histogram; ±SEM; n = 3; P values are relative to the control). Addition of control monoclonal antibody to mitochondrial HSP70 had no effect on any of these processes (data not shown). Bar, 10 μm.