TIMPs 2, 3, and 4 and siRNA suppression of MT1-MMP block EC motility in 3D collagen matrices but not on 2D collagen substrates. (A-C) For 2D assays, nuclear GFP-labeled ECs were seeded on collagen coated plastic while for 3D assays, nuclear GFP-labeled ECs were placed into collagen gels. Time-lapse fluorescence microscopy was used to track cell motion using nuclei as a measure of EC migratory events. GM6001 was added to the culture media at 5 μM while the TIMPs were used at 5 μg/mL in the media. siRNA knockdown was performed in ECs for MT1-MMP, MMP-1, and Luciferase as control. n = 25 cells quantitated for each condition. P < .01 compared with control. (A) Representative overlays of tracking data are shown. The images show the movement of single cells after siRNA treatment on 2D collagen surfaces versus within 3D collagen matrices. Bar equals 100 μm.