Adhesion, migration and invasion assays

For adhesion assays, HUVEC cells (75,000 cells/well) were cultured in 96-well plates covered with fibronectin (2.5 μg/ml) and collagen 0.05 μg/ml at 4 °C for 10 min. Adhesion was then allowed at 37 °C during 30 min in the absence or presence of plocabulin. After 3 washes with PBS, remaining cells were fixed with glutaraldehyde (0.1%) and stained with sulforhodamine B. As positive and negative controls, we have used MnCl₂ (0.5 mM), latrunculin A (3 μM) and cytocalasin D (2 μM), respectively. For migration and invasion assays, 6.5 mm-diameter transwell chambers (Sigma-Aldrich, St. Louis, MO, USA) with polycarbonate membrane (pore size 8.0 μm) were used. HUVEC cells (1.5 × 105) were seeded on the upper compartment of the transwell membrane in 100 μl of serum-free culture medium containing or not plocabulin. The lower compartment (well) was filled with 600 μl of serum-free culture medium containing or not a chemoattractant (FBS 2%). For the invasion assays, the upper compartment of the transwell was previously coated with 12 μg of matrigel basement membrane matrix to create a physical barrier between the two compartments of the chamber. After 24 h of incubation, culture medium was removed from the inside of the chamber, and the non-migrated cells on the upper side of the membrane were wiped off using a cotton swab. Migrated cells on the lower side of the membrane were fixed with a glutaraldehyde 1% solution, washed and stained with sulforhodamine B following standard techniques.