Ro 32 0432

Scratch wound migration assays were performed on confluent monolayers of AGS cells as previously described [41 (link)]. Transwell migration and invasion chemotaxis assays were performed using BD inserts (Corning, NY, USA) as previous described (25,000 cells per insert) [46 (link)]. Chemerin or CAM-conditioned medium (CM) were added in the lower well together with CCX832, α-NETA, Ro-320432, human recombinant TIMP-1 or TIMP-2 (R&D Systems), or vehicle, as appropriate.

1 × 10⁵ calcein-AM labelled CD11b+ cells were incubated on 48-well culture plates containing endothelial cell monolayers (HUVEC) or coated with 5 μg/ml recombinant soluble VCAM-1 (R&D Systems), ICAM-1 (R&D Systems), vitronectin, or collagen for 20 min at 37 °C in basal media, culture media from Lewis lung carcinoma cells (TCM) or DMEM containing 200 ng/ml SDF1α, IL-1β, IL-6, TNFα, or VEGF-A (R&D Systems) without or with 1 µM inhibitors directed against MLCK (ML-7 or MW01-022AZ), PKC (Ro-32-0432), calcium/calmodulin (W7), or myosin (Blebbistatin). Additionally, CD11b+ myeloid cells from WT, Rap1^−/−, p110γ^−/−, and Mlck210^−/− mice as well as Mlck, Myh9, Myh10, and non-silencing siRNA-transfected and RasV12, RapV12, and p110γCAAX-transfected cells were similarly incubated for 20 min on 5 μg/ml rsVCAM-1. After washing three times with warmed medium, adherent cells were quantified using a plate fluorimeter (TECAN). Serum-free Lewis lung carcinoma conditioned medium was prepared by culturing LLC cells in DMEM for 24 h and centrifuging culture medium at 10,000 × g to remove debris.