Regulation of MT1-MMP–positive endosome positioning by ARF6 and JIP3/JIP4. (A) JIP4 staining of MT1-MMPmCh–expressing MDA-MB-231 cells. Bars: 5 µm; (insets) 2 µm. (B and C) Mean invasion area of multicellular spheroids after 2 d in 3D collagen normalized to mean spheroid area at T0 and to invasion of siNT-treated spheroids set to 100 ± SEM. n, spheroids number. ***, P < 0.001. (D) Collagenolysis by MDA-MB-231 cells treated with the indicated siRNAs. Values are mean normalized degradation index ± SEM. n, number of cells analyzed for each cell population. *, P < 0.05; ***, P < 0.001 (as compared with siNT-treated cells). (E) Distribution of MT1-MMPmCh endosomes in MDA-MB-231 cells plated on 2D gelatin. Mean percentage of MT1-MMP–positive endosomes according to their cell center-to-cell periphery position ± SEM. **, P < 0.01; ***, P < 0.001 (compared with siNT distribution). (F) Inverted still images from time-lapse sequences of MDA-MB-231 cells expressing MT1-MMPmCh treated with indicated siRNAs (see Video 1). Insets show color-coded time projections of selected time frames from these sequences (color code shown on the left of inset 1) corresponding to the boxed regions at higher magnification. Bars: 10 µm; (insets) 5 µm. (G) Distribution of MT1-MMPmCh endosomes in MDA-MB-231 cells expressing the indicated JIP3-LZI construct as in E. (H) MT1-MMPmCh endosome distribution as in E. *, P < 0.05; ***, P < 0.001 with siARF6+sip150^Glued as compared with siARF6 treatment and siJIP3/4+sip150^Glued as compared with siJIP3/4. ns, not significant.