*4.13. Matrigel-Invasion Assay*

The ability of cells to migrate or invade through a Matrigel barrier was measured in a Boyden chamber assay with polycarbonate membranes. After 48 h of siRNA knockdown, an equal number of 12Z cells (2 <sup>×</sup> <sup>10</sup><sup>4</sup> ) were re-suspended in 100 µL of growing media supplemented with 1% *v*/*v* FCS and antibiotics and plated on top of matrigel coated filter (Corning Matrigel growth factor reduced (354230); Corning Incorporated, Corning, NY, USA, 1% matrigel solution in PBS, filter: 6.5 mm diameter, 8 µm-pores, Corning Incorporated, Corning, NY, USA). The cells were allowed to migrate/invade for 12 h toward the bottom of the well, which contained media supplemented with 10% FCS. Cells on the lower surface of the filter were fixed with 4% PFA and stained with CyQUANT™ Direct Red nucleic acid stain (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and photographed with an ×10 objective under the microscope. The values for invasion were taken as the average number of invaded cells per photographic field over five independent fields per experiment and expressed as averages of triplicate experiments.
