ECM degradation assay using DQ™-collagen IV

Three-dimensional ECM (40 μl) consisting of Matrigel (10 mg/ml) and DQ™-collagen type IV (2.5 mg/ml, Life Technologies) at a volume ratio of 10:1 was overlaid onto a Lab-Tek 8 chamber borosilicate cover glass system. After a 30 min incubation at 37°C, SNU449_Cp or SNU449_Tp cells (35,000 cells/condition) were overlaid onto the ECM. After another 30 min incubation at 37°C, each group was overlaid with 300 μl of complete medium containing 10% Matrigel and either DMSO (Control) or ARP-100 (50 μM). The cells were then incubated for 20-24 h, and DIC and fluorescence images were randomly obtained using the Nikon Plan Apochromat 40×/0.95 N.A. objective of a Nikon Eclipse Ti microscope (Nikon, Japan).