The LLY motif in the MT1-MMP C terminus is crucial for the recovery of regular podosome densities during podosome reformation. Statistical evaluation of podosome density (podosomes/100 µm2) during a podosome reformation assay. Parameters were evaluated in cells before disruption of podosomes (“-PP2”) or after treatment with podosome-disrupting PP2 and washout of the drug for the indicated periods, in cells treated with control siRNA (gray) or with MT1-MMP-specific siRNA (red). Subsets of MT1-MMP–depleted cells were also expressing a construct of the MT1-MMP C terminus that is not targeted by the siRNA (ΔNterm; green) or of a full-length MT1-MMP construct mutated in the LLY motif, which was rendered siRNA insensitive (LLY/AAA insens.; blue). n = 3 × 30, for cells from three different donors. Note that podosomes are mostly absent at 0 min of the washout; therefore, podosome density was not evaluated for this time point. Note also that expression of the ΔNterm constructs rescues the recovery of regular podosome density, whereas expression of the LLY/AAA mutant construct does not. Values are given as means ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. For dot diagrams of podosome numbers, podosome area, and podosome density, see Fig. S6. For specific values, see Table S2.
