MT1-MMP mediates hMSC-dependent collagenolytic activity. (A) siRNA-dependent silencing of MMP expression in hMSCs as assessed by RT-PCR. hMSCs were electroporated with 40nM scrambled (Scr siRNA) or MMP-1–, MMP-2–, MMP-13–, MT1-MMP–, or MT3-MMP–specific siRNAs and cultured in growth medium for 2 days followed by RT-PCR analysis of MMP expression with GAPDH as loading control. (B-C) hMSCs electroporated with each of the respective siRNAs or cotransfected with a control expression vector (MT1siRNA + Vec) or mouse MT1-MMP expression vector (MT1siRNA + mMT1) were seeded atop Alexa Fluor 594–labeled type I collagen film (red) for 4 days and collagenolytic activity quantified by hydroxyproline release (mean ± SEM; n = 3. *P < .05) as well as visualized by confocal laser microscopy. Bar, 50 μm. (D) Cells electroporated with Scr or MT1 siRNA were incubated on Alexa Fluor 647–labeled collagen (blue) for 2 days, and cell surface was stained with anti–MT1-MMP (red). Colocalizations of MT1-MMP “dots” on the basal cell surface with areas of collagen degradation were found in Scr siRNA–treated cells. Neither MT1-MMP nor zones of degraded collagen were found in MT1 siRNA–treated cells. The position of a cell body overlying the collagen substratum is outlined by the dotted white lines. Bar, 10 μm. (E) 3D reconstructions of collagen (Alexa Fluor 647 labeling, blue), phalloidin (green), and cell surface-localized MT1-MMP (red). Black arrows mark the direction from top to bottom. White lines mark either the yellow dots (comprised of MT1-MMP–positive areas that colocalize with phalloidin), which appear in the zones of collagen degradation in Scr siRNA-cells (left), or green dots (ie, MT1-MMP–negative and phalloidin-positive), which appear atop the intact collagen surface in MT1-MMP siRNA-silenced cells (right). Bar, 10 μm.