Rat tail collagen 1

Cells were serum starved and treated with 1 ng/ml of mitomycin (Sigma M4287) for 12 h to inhibit cell division. Upper chambers (8 μm pores, Becton Dickinson, USA) cell chambers were placed in 24-well format transwell plates. Starved cells were plated in assay media (200 μl) (20 000 cells/well in the upper chamber). Lower chambers were pre-coated for 2 h with rat tail collagen I (Roche). To initiate migration, media containing 10% serum (600 μl) was placed in the lower chamber as attractant. After 48 h of incubation, cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and stained with 0.5% crystal violet for 15 min at room temperature. Inserts were washed with PBS and air dried for 15 min. Cells on the upper side (non-migrating) of the filter were removed by swabbing with cotton wool. Migrating cells were imaged on a Zeiss Axioskop 2 fluorescence microscope using a 10x objective (Zeiss, NA 0.3) and a Spot RT/SE CCD camera (Diagnostics) in mosaic format. Cells were counted using ImageJ software cell counter.

Eribulin mesylate (Eisai, Research Triangle Park, NC), paclitaxel and vinblastine (Sigma-Aldrich, St Louis, MO) were dissolved in DMSO and stored as a 5 mM stock in glass vials at −20°C. Serial dilutions of drugs were performed in 50% DMSO in glass vials and stored as 100-fold stock at −20°C. Wells of 96-well plates were coated with 25 μg/ml rat tail collagen I (Roche Diagnostics, Mannheim, Germany) in PBS, prior to seeding with 5000 cells per well. Effect of the drugs on cell viability was assessed with a sulforhodamine B assay [37 (link)] after 72 h of drug exposure. The final concentration of DMSO (0.5%) was the same for both drug-treated and control cells.