Ultrapure agar

For proliferation assays, 24 h after transfection, 0.5X10⁴ cells were plated in 6-well plates in triplicate, and cells were quantified in a cell counter (Nexcelom) at the indicated times. The transwell cell migration assays were performed in duplicate, using 6.5 mm diameter Falcon cell culture inserts (8 mM pore size, Falcon) in 24 well cell culture plates. 1X10⁵ cells in serum-free media were transferred to the upper chamber, while the lower chamber contained media with 10% FBS. Following overnight incubation, the cells remaining on the upper surface were removed with a cotton swab. Migrated cells were fixed, visualized microscopically, photographed, and quantified using Image J. For soft agar assays, 1X10⁵ stably transfected cells were mixed with complete media containing 0.4% of ultrapure agar (Invitrogen) and placed over 0.6% basal agar in 60 mm dishes. 3 to 5 plates per transfectant were used. Cells were grown for 3–4 weeks, and colonies were stained with Nitrotetrazolium Blue Chloride (1 mg/ml), photographed by microscopy, and quantified with a colony counter.