In Vitro Invasion Assay Protocol

In vitro invasion assays were performed as previously described^{59 (link)}. In brief, 1.5 × 10⁵ cells were plated in the top wells of Growth Factor Reduced Matrigel-coated invasion chambers (8 µm pore size, BD Bio Coat). Media containing 5% serum was added to the lower chamber, and cells were allowed to invade along the serum gradient for 18 h at 37 °C. The assay was fixed with 3.7% PFA for 20 min and stained with NucBlue (Invitrogen) to visualize the nuclei. When siRNA-transfected cells were used, siGLO-Red (Dharmacon, Lafayette, CO, USA) was co-transfected in the cells to identify siRNA-treated cells. The membrane was detached from the chamber and mounted on a coverslip, and 10 random fields of view were imaged across the membrane at 20× magnification on an IX81-ZDC microscope (Olympus, Tokyo, Japan). The number of invading cells was counted manually with ImageJ software by thresholding onto the nucleus, and data are reported as the means of 3 experiments for each condition.