ADAMTS-1 knockdown increased invadopodia formation in MDA-MB-231 cells. Cells were grown on gelatin-Alexa-567 for 6 hours. Image A is a merged image of the blue (nuclei stained with DAPI), red (gelatin-Alexa 567) and green (ADAMTS-1 shRNA-GFP) channels. Z-projections of 10 optical sections together with orthogonal projections onto XZ (top) and YZ (left) planes were generated (B). The white lines in (B) indicate points of the XY image projected to generate orthogonal (perpendicular) XZ (top) and YZ (right) planes. Orthogonal projections of XZ and YZ illustrate digestion at the ventral surfaces of cells. Substrate digestion could be clearly observed when planes XY, XZ and YZ were viewed in the same volume (C and D, arrowhead). Images C and D were rotated to observe the cell’s ventral surface. The control samples (treated with non-silencing shRNA-GFP) show discrete matrix digestion (E and F). The area measurements demonstrate that ADAMTS-1 knockdown significantly increased matrix digestion (G). Digested areas in close proximity to the cell ventral surfaces indicate that ADAMTS-1 knockdown induced invadopodia activity. The asterisks indicate significant data in comparison to the control (*p < 0.05, ***p < 0.001). A total of 10 random fields were imaged per experimental group. Scale bar: 10 μm.