Figure 4

RhFNHN29 and rhFNHC36 inhibit MHCC97H cell adhesion to FN substrate and migration toward FN. (A), MHCC97H cell suspension (2 × 10⁵/100 μl) with or without Mn²⁺ (0.1 mM) was added to a 96-well plate coated with either BSA, type I collagen (20 μg/ml), type IV collagen (20 μg/ml) or FN (20 μg/ml). (B), Effects of different antibody concentrations (6, 12, 25 and 50 μg/ml) on MHCC97H cell adhesion to FN. (C), MHCC97H cell suspension (2 × 10⁵/100 μl) containing Mn²⁺ (0.1 mM) was pretreated with or without 25 μg/ml control IgG and function-blocking mAb to αv (P2W7), β3 (BV4) or β1 (8A2), respectively, and then seeded to a 96-well plate coated with FN (20 μg/ml) or poly-L-Lys (20 μg/ml) in the presence or absence of FN, rhFNHN29 and rhFNHC36 (50, 100 and 200 μg/ml), respectively. The data represent the mean ± SD of three determinations. (D), A cell invasion assay was carried out in blind-well chambers. MHCC97H cell suspension (5 × 10⁴/100 μl) containing Mn²⁺ (0.1 mM) treated with or without FN, rhFNHN29 and rhFNHC36 (50, 100 and 200 μg/ml), and control IgG, or anti-αv (P2W7), -β3 (BV4) or -β1 (8A2) (25 μg/ml) were incubated at 37°C for 1 h and then added to the top chambers, respectively. After incubation for 48 h, the number of cells migrating across the Matrigel-coated filter into the bottom chamber was counted. The data represent the mean ± SD of three determinations. (E), Compared with the morphous of MHCC97H cells under normal conditions (×200 magnification), representative pictures for migrated MHCC97H cells on the bottom chamber (×100 magnification) in the presence of FN, rhFNHN29 and rhFNHC36 (200 μg/ml) showed a fibroblastic appearance.