Cell adhesion assays

Primary bone marrow-derived macrophages were obtained from femoral bone marrow suspensions of C57BL/6 mice and plated at 3×10⁶ cells/mL and differentiated for 7 d in the DMEM medium with 10 % FBS and 5×10^-3 mg/mL murine G colony-stimulating factor (Pepro Tech, no. A2714). Mouse RAW 264.7 cells were cultured in DMEM medium supplemented with 10 % FBS and 1% penicillin/streptomycin (100 U/100 mg) solution (Life Technologies, Grand Island, NY).

For macrophage adhesion to VSMCs, VSMCs grown on glass coverslips in 6-well plates were transfected with Sal-miR-1 and 3 for 6 h, and then treated with thrombin for 24 h. Then the RAW264.7 cells or primary bone marrow-derived macrophages were added to each hole, and incubated at 37 °C for 30 min. The coverslips were washed with fresh serum-free medium 3 times for 15 min each, and the non-adherent macrophages were washed out. The coverslips were collected, and 4% paraformaldehyde was added and fixed for 10 min. Immunofluorescence staining for VSMCs using anti-SM α-actin (rabbit polyclonal, 1:100, 14395-1-AP, Proteintech), macrophage using anti-MAC-2 (mouse monoclonal, 1:50, 60207-1-Ig, Proteintech) was performed. At least five random fields in each coverslip were observed, and the number of cells was counted.