Integrin functions are impaired on RIAM^−/− macrophages. (A) Vinculin (red) and F-actin staining (phalloidin; blue) of wt and RIAM^−/− BM-derived DCs expressing EGFP or RIAM-EGFP (green). Scale bar represents 5 μm. (B-E) Static adhesion assay of BM-derived wt, RIAM^−/−, and talin-1^−/− macrophages to FN (B), VN (C), VCAM-1 (D), and ICAM-1 (E) in the presence or absence of 0.5 mM MnCl₂ relative to wt macrophages (n ≥ 5 independent experiments). Adhesion was measured indirectly using crystal violet staining and optical density (OD) measurement at 595 nm. (F) Representative images of macrophages seeded for 30 minutes on FN, VN, or VCAM-1 and on ICAM-1 in the absence or presence of 0.5 mM MnCl₂. Scale bar represents 50 μm. (G) Mean ± SD values of spreading area of wt, RIAM^−/−, and talin^−/− macrophages seeded for 30 minutes on FN, VN, VCAM-1, or ICAM-1 (n = 5 independent experiments with 50 cells measured per experiment). (H) Mean ± SD values of spreading area of wt and RIAM^−/− macrophages seeded for 30 minutes on ICAM-1 in the absence or presence of 0.5 mM MnCl₂ (n = 3 independent experiments with 50 cells measured per experiment). (I-J) wt and RIAM^−/− macrophages were either kept in suspension or plated for 20 minutes on FN or ICAM-1, and analyzed for global tyrosine phosphorylation (I) and for phosphorylation and total expression of Src, FAK, and Pyk2 (J). GAPDH served as loading control. (K) wt and RIAM^−/− macrophages were either kept in suspension or plated for 20 minutes on FN or ICAM-1 in the presence of 0.5 mM MnCl₂ and analyzed for Pyk2 phosphorylation. P values indicate significant differences and were calculated with the Student t test. sus., suspension.