Knockdown of MT1F and MT1M Impacts Cell Invasion

Specific siRNA was used to knockdown MT1F and MT1M expression (Thermo Scientific). MCF7 (approximately 50,000 cells) was transfected with MT1F and MT1M siRNA seeded onto the top insert (layered with Matrigel) of an invasion chamber (BD Biosciences, Franklin Lakes, NJ, USA). The invasion chambers were then incubated at 37°C in 5% CO₂ for 20 h. Cells that did not invade through the Matrigel (on the upper surface of the insert membrane) were mechanically removed with cotton tip applications and several washes with PBS. Invaded cells on the bottom of the coated membranes were visualized using a fluorescence microscope with a × 20 objective after incubation with Hoechst stain (Life Technologies). Images were obtained from four standardized non-overlapping fields. Invaded cells were counted using the Image J software (http://rsbweb.nih.gov/ij/). Invasion assays were done in triplicate; images of four fields per well (covering about 85% of the well) were taken for counting invaded cells. Cellular proliferation was assayed using CellTiter-Glo® Luminescence Kit (Promega, Madison, WI, USA) according to the manufacturer’s directions as described earlier [43 (link)].