Figure 5. Cell adhesion and migration in response to the N-glycosylation of human and porcine fibronectin.

(A) Cells were pre-incubated with serum-free medium containing H₂O (control), serum-free medium containing 0.5 mM EDTA, or culture medium containing 2 mM MnCl₂ before plating on the indicated concentrations of fibronectin [FN] treated with (+) or without (-) PNGase F. Cells were plated on [FN] for 10 min and then their attachment was measured. Percentage of cells remaining attached on [FN] was shown. Data are the mean ± s.e.m. (n = number of independent experiments). *p < 0.05; **p<0.01; ***p < 0.001; NS, no significance. (B) Cell lysates from U2OS cells plated on 10 μg/ml FN treated with (+) or without (-) PNGase F for 10 min or 16h (overnight) were analyzed by Western blotting using antibodies against FAK, FAKpY397, total integrin β1 (CD29), activated integrin β1 (B44) and α-tubulin. Bottom: ratio indicates the expression of FAKpY397 normalized against FAK and activated integrin β1 (B44) against to total integrin β1 (CD29) from Western blotting. The ratio in the indicated conditions is calculated relative to that in the first lane in the same gel. Data are the mean ± s.e.m. (n = 5 independent experiments). NS, no significance. (C) Homo and porcine plasma fibronectin with (+) or without (-) α2-3,6,8 Neuraminidase (Sialidase) digestion were analyzed to assess the spectral counts of N-glycosylated residues with or without sialic acids at the various individually detected N-glycosylation sites. The plot shows the percentage of the spectral counts of the N-glycosylated residues with or without sialic acids relative to the total spectral counts for all detected N-glycosylation sites. The results demonstrated an efficient process for the trimming of sialic acids across the various N-glycosylation sites. (D) Cells in serum-free medium were plated for 20 min on [FN], treated with (+) or without (-) α2-3,6,8 Neuraminidase (Sialidase). The fold of cells remaining attached on [FN] relative to that on 0 μg/ml fibronectin is shown. Data are the mean ± s.e.m. (n = number of independent experiments). NS, no significance. (E) Cells in serum-free medium were seeded on plates coated with [FN], accompanied by ddH₂O, AAL or WGA for 20 min. The fold of cells remaining attached on [FN] relative to that on 0 μg/ml fibronectin was measured. Data are the mean ± s.e.m. (n = number of independent experiments). *p < 0.05; NS, no significance. (F) Cells were plated on homo or porcine fibronectin, treated with (+) or without (-) PNGase F and then assayed for wound-healing migration. The percentage of wound closure at 10-h time point of wound-healing migration was calculated using Metamorph software and plotted. Data are the mean ± s.e.m. [homo: n = 5 (-PNGase F), n = 6 (+PNGase F) independent experiments; porcine: n = 6 (-PNGase F), n = 5 (+PNGase F) independent experiments]. **p<0.01; ***p < 0.001.