Migration and invasion assays

MDA-MB-468 cells were pretreated for 72 hrs with doxycycline (IC₅₀ concentration of 11.39uM) or vehicle. On day 4, cells were removed and counted with Trypan blue (Corning, USA). For the migration assay 30×10⁴ viable cells were seeded on the upper layer of non-coated membrane trans-well inserts (pore size, 8.0um, Merck Millipore). Cells were allowed to adhere in starvation medium overnight and the next day the inserts were placed into the lower chambers filled with DMEM (10% FBS) incubated for 24h at 37°C at 5% CO₂ atmosphere. 24 hours later cells on the top of the insert were carefully removed with a cotton swab. The invasion assay was performed as above, but Matrigel-coated membranes were used instead. Thereafter, the inserts were removed and the non-invading cancer cells remaining on the upper layer were scraped off. Cells that had migrated or invaded the matrigel and subsequently migrated to the bottom of the transwell were fixed with 3.7% paraformaldehyde for 30min, stained with 1% crystal violet in 2% ethanol for 30min, then viewed under a Nikon inverted light microscope and photographed. Three biological repeats were performed for each assay.