Src sirna

Boyden chambers were used for transwell migration and invasion assays. Cells were serum-starved for 24 h and 5 × 10⁴∼3 × 10⁵ cells were seeded with serum-free medium into the top of the transwell inserts with 8 μm pore size for the migration assay, or into the top of the transwell coated with matrigel (BD Biosciences) for the invasion assay. In the bottom chamber, regular medium containing serum was added. To facilitate the migration of MCF7, MDAMB361, or T47D cells, NIH3T3 cells were seeded in the bottom chamber as a chemo-attractant. For the Dox-inducible TLK2 overexpression model, 0, 50, 100, or 200 ng ml⁻¹ of Dox was administered for 2 weeks before the migration and invasion assay. To verify the dependence of migration and invasion properties on TLK2 expression, Dox was withdrawn for 4 days following 2 weeks of Dox treatment to deplete the excess TLK2 protein. To observe the effect of SRC, EGFR or FAK inhibition on TLK2-driven cell motility, 20 nM of siRNA targeting EGFR and FAK or 20–40 nM of SRC siRNA (Dharmacon) were transfected for 3 days before perform the transwell migration assay. After 48–72 h, the inserts were fixed in 4% formaldehyde and stained with hematoxylin and eosin. The migrated and invaded cells were counted by GelCount colony counter (Oxford Optronix Ltd.).