Figure 3: VAMP3 depletion disrupts association between MT1-MMP and CD9.

(a) Lysates from control-shRNA- or VAMP3-shRNA-electoporated cells were immunoprecipitated using polyclonal anti-CD9 antibody. Total cell lysates (left) and immunoprecipitates (right) were resolved by SDS–PAGE, transferred to polyvinylidene difluoride membrane and probed as indicated. Blots shown are representative of more than four independent immunoprecipitation replicates. (b) LOX cells and those expressing shRNA, as indicated, were incubated with mouse anti-MT1-MMP (lanes 1, 4 and 5) or control mouse IgG (lane 3) as described in Methods. These cells were incubated for 16 h in pre-cleared complete culture media before shed microvesicles were isolated. Microvesicle lysate was then resolved by SDS–PAGE and analysed by western blotting as indicated. (c) LOX cells expressing either scrambled-shRNA or VAMP3-shRNA were embedded in BD Rat Tail collagen I matrix and imaged 24 h post embedding, to allow for shRNA expression. Frames shown are representative stills captured approximately every 20 min. Scale bar, 25 μm. Control (d) or NSC405020-treated (e) LOX cells were plated on FITC-gelatin and allowed to invade overnight. Coverslips were fixed and stained as indicated, to examine the extent of cell invasion. Scale bar, 25 μm. (f) Cells expressing scrambled siRNA (top) or MT1-MMP siRNA (bottom) were allowed to invade TRITC-conjugated matrix overnight. The cells were then fixed, stained as indicated and analysed by confocal microscopy, to examine invasive capacity. Scale bar, 25 μm. (g) LOX cells transfected to express MT1-MMP-siRNA (top) or scrambled siRNA (bottom) were embedded in a layer of rat tail collagen-I and allowed to invade. Cell invasion was monitored by phase-contrast microscopy over the course of 12 h. Stills represent sequential images every 90 min. Scale bar, 25 μm.