Cell migration assays were performed for 24h as previously described (Forsyth et al., 2001). 1 × 105 cells (MDA-MB-231) in serum-free media (± alcohol ± inhibitors) were added to the upper well of PET membrane transwells (12 well/8μM pore; Falcon, Becton Dickinson, Franklin Lakes, NJ). TNF-α (100ng/ml) was then added to the lower well. For some wells ethanol (E, 0.2%, 43 mM) was added before adding cells ± specific inhibitors of the EGFR (AG1478, 500μM); TACE (TAPI-2, 20μM); or ERK1/2 (MEK)(PD98059, 30μM). Assay was stopped by removing cells from the upper well with a cotton swab and inserts fixed and stained with crystal violet to visualize cells. Data are mean ± SE. cells per high power field (40x objective) from 5 fields per insert for duplicate wells in a representative from three experiments.
