Transwell Invasion and ECM Degradation Assay

Transwell invasion assay was performed as described previously^{4 (link)}. In brief, cells were loaded onto the upper well of the Transwell chamber with 8 µm ϕ pore membrane (Coster), precoated with Matrigel on an upper side of the chamber. The lower well was filled with 600 µl of DMEM containing 10% FBS. After incubation for 24 hr, cells invaded to lower surface of the membrane were counted. For ECM degradation assay, glass coverslips were coated with gelatin conjugated with either Alexa Fluor 594 (Invitrogen) (Alexa-gelatin) or fluorescein (Invitrogen) (FL-gelatin) as described^{65 (link)}. Transfected cells were trypsinized, replated on these glass coverslips, and cultured for 6 hr. After fixation, cells were fixed and stained with phalloidin. Number of invadopodia, identified as F-actin dots in the areas of degraded gelatin, was quantified by using the ImageJ particle analysis tool.