SNX27 carries MT1-MMP to the cell surface and contributes to its recycling. (A) Matrigel invasion assay was performed (as described earlier) in MDA-MB-231 cells transfected with control siRNA and siRNA targeting indicated molecules. The number of invasive cells was counted. One-way ANOVA, *** P < 0.001. The graph represents means ± SEM. (A′) Cells depleted for SNX27 were fixed after 72 h and immunostained with Tks5. Phalloidin was used to label actin. Images were acquired with a confocal microscope and the number of Tks5/actin (invadopodia) per cell were counted (N = 3, n = 30; scale bars = 10 µm, inset = 4 µm). The insets display the Tks5/actin-rich structures. One-way ANOVA, *** P < 0.001. The graph represents means ± SEM. (B) Cells were treated with siRNA against SNX27 and 60 h after treatment were transfected with GFP vector or GFP-SNX27WT. After 12 h, cells were fixed and imaged using a confocal microscope (N = 3, n = 60; scale bars = 10 µm, inset = 4 µm). The insets show the degradation spots. (C) MDA-MB-231 cells were transfected with control siRNA and siRNA targeting SNX27. 60 h after transfection, pHluorin–MT1-MMP and pHluorin–MT2-MMP were transfected. Cells were allowed to adhere to the gelatin-coated imaging dishes for 6 h and then subjected to TIRF microscopy. The number of vesicles and their total area was quantified and plotted. (N = 3, n = 30; scale bars = 10 µm). Two-tailed Student’s t test, **P < 0.01, *** P < 0.001. The graphs represent means ± SEM. (D) Cells were treated with control and SNX27 siRNA and subjected to biotinylation to measure only the surface MT1-MMP levels. Cells were labeled with noncleavable biotin and lysed, followed by binding with Neutravidin beads (refer to Materials and methods). The graph represents the quantification from the three individual blots for surface MT1-MMP levels (N = 3; additional blots in Fig. S5). Two-tailed Student’s t test, * P < 0.05. (E) Cells were treated with control and SNX27 siRNA. After 72 h, cells were incubated with antibodies against MT1-MMP at 4°C for 1 h, then shifted to 37°C for 15 min. Cells were fixed, stained with DAPI, and imaged with a confocal microscope. The total intensity of the MT1-MMP vesicles per frame was calculated and plotted (N = 3, n = 3,000; scale bars = 10 µm, inset = 4 µm). The insets show the MT1-MMP antibody uptake by the cells. Two-tailed Student’s t test, **** P < 0.0001. The graph represents means ± SEM. (F) MDA-MB-231 cells were cotransfected with GFP-SNX27 and mCherry–MT1-MMP and 12 h after transfection were imaged by a live confocal microscope (N = 3, n = 25; scale bars = 5 µm, inset = 4 µm). The insets represent various events from different time points of the live-cell imaging, where SNX27 was associated with MT1-MMP vesicles. (G) MDA-MB-231 cells transfected with mCherry–MT1-MMP and GFP-SNX27 were subjected to TIRF microscopy, and endosome dynamics near the cell surface were analyzed using a TIRF microscope. The inset panel represents a series of frames (along with the time point) from a captured video (inset scale bars = 5 µm). Arrowheads show vesicles positive for SNX27 and MT1-MMP near the cell surface (N = 3, n = 12). N, number of experimental repeats; WCL, whole cell lysate.
Figure 4.

SNX27 carries MT1-MMP to the cell surface and contributes to its recycling. (A) Matrigel invasion assay was performed (as described earlier) in MDA-MB-231 cells transfected with control siRNA and siRNA targeting indicated molecules. The number of invasive cells was counted. One-way ANOVA, *** P < 0.001. The graph represents means ± SEM. (A′) Cells depleted for SNX27 were fixed after 72 h and immunostained with Tks5. Phalloidin was used to label actin. Images were acquired with a confocal microscope and the number of Tks5/actin (invadopodia) per cell were counted (N = 3, n = 30; scale bars = 10 µm, inset = 4 µm). The insets display the Tks5/actin-rich structures. One-way ANOVA, *** P < 0.001. The graph represents means ± SEM. (B) Cells were treated with siRNA against SNX27 and 60 h after treatment were transfected with GFP vector or GFP-SNX27WT. After 12 h, cells were fixed and imaged using a confocal microscope (N = 3, n = 60; scale bars = 10 µm, inset = 4 µm). The insets show the degradation spots. (C) MDA-MB-231 cells were transfected with control siRNA and siRNA targeting SNX27. 60 h after transfection, pHluorin–MT1-MMP and pHluorin–MT2-MMP were transfected. Cells were allowed to adhere to the gelatin-coated imaging dishes for 6 h and then subjected to TIRF microscopy. The number of vesicles and their total area was quantified and plotted. (N = 3, n = 30; scale bars = 10 µm). Two-tailed Student’s t test, **P < 0.01, *** P < 0.001. The graphs represent means ± SEM. (D) Cells were treated with control and SNX27 siRNA and subjected to biotinylation to measure only the surface MT1-MMP levels. Cells were labeled with noncleavable biotin and lysed, followed by binding with Neutravidin beads (refer to Materials and methods). The graph represents the quantification from the three individual blots for surface MT1-MMP levels (N = 3; additional blots in Fig. S5). Two-tailed Student’s t test, * P < 0.05. (E) Cells were treated with control and SNX27 siRNA. After 72 h, cells were incubated with antibodies against MT1-MMP at 4°C for 1 h, then shifted to 37°C for 15 min. Cells were fixed, stained with DAPI, and imaged with a confocal microscope. The total intensity of the MT1-MMP vesicles per frame was calculated and plotted (N = 3, n = 3,000; scale bars = 10 µm, inset = 4 µm). The insets show the MT1-MMP antibody uptake by the cells. Two-tailed Student’s t test, **** P < 0.0001. The graph represents means ± SEM. (F) MDA-MB-231 cells were cotransfected with GFP-SNX27 and mCherry–MT1-MMP and 12 h after transfection were imaged by a live confocal microscope (N = 3, n = 25; scale bars = 5 µm, inset = 4 µm). The insets represent various events from different time points of the live-cell imaging, where SNX27 was associated with MT1-MMP vesicles. (G) MDA-MB-231 cells transfected with mCherry–MT1-MMP and GFP-SNX27 were subjected to TIRF microscopy, and endosome dynamics near the cell surface were analyzed using a TIRF microscope. The inset panel represents a series of frames (along with the time point) from a captured video (inset scale bars = 5 µm). Arrowheads show vesicles positive for SNX27 and MT1-MMP near the cell surface (N = 3, n = 12). N, number of experimental repeats; WCL, whole cell lysate.

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