Fig. 4

rCD248 interferes with melanoma cell adhesion and migration. A, B Cell adhesion assay. B16F10 cells were allowed to adhere to BSA- or fibronectin (FN)-coated well with or without rCD248 (1 µM rCD248D1-4) for 30 min and followed by crystal violet stain. A Representative images of cell adhesion to fibronectin. B Statistical analysis thereof. N = 4. ***P < 0.001. C, D Western blot analysis of FAK activation (phosphorylation of Tyr576 FAK, p-FAK(Tyr576) in cells after cell adhesion assay. N = 4. ***P < 0.001. B16F10 cells were subjected to (E and F) wound recovery migration assay and G–J chemotactic migration assay. E Representative images of wound recovery assay and statistical analysis thereof (F). B16F10 cells were treated with rCD248D1-4 (1 µM) or vehicle control (Ctrl) and photographed at various time points. N = 4. **P < 0.01; ***P < 0.001. G, H Boyden chamber migration assay of B16F10 cells pretreated with various concentrations of rCD248D1-4 protein 1 h before being applied to the upper well of a Boyden chamber and with the fibroblast-cultured conditioned medium (CM) as chemoattractant. Migrated cells were stained G and enumerated H 3 h after migration. N = 5. ***P < 0.001 compared with vehicle control. I, J Boyden chamber migration assay of B16F10 cells pre-treated with 1 µM of various rCD248 proteins (rCD248D1, rCD248D3, and rCD248D1-4) for 1 h before being applied to the upper well of a Boyden chamber and with the CM at the bottom well as chemoattractant. I Representative images of Boyden chamber migration assay. Migrated cells were stained and enumerated 3 h after migration. J The statistical analysis of Boyden chamber migration assay of B16F10 cells treated with different rCD248 proteins. N = 5. ***P < 0.001 compared with control (Ctrl). HEK293 cells transfected with GFP-tagged CD248 or with GFP as control were subjected to K western blot analysis of protein expression and L, M Boyden chamber migration assay. L Representative images of Boyden chamber migration assay in HEK293 cells with and without rCD248D1-4 (1 µM) treatment. Migrated cells were stained and enumerated 4 h after migration. M Statistical analysis of Boyden chamber migration assay of HEK293 cells. N = 6. ***P < 0.001