SNX27 and retromer reside on the endosomal subdomains of MT1-MMP to facilitate its recycling and matrix degradation activity. (A) MDA-MB-231 cells expressing GFP–MT1-MMP were fixed, immunostained with an antibody against SNX27 and Vps26, and analyzed by super-resolution microscopy 3D-SIM. Boxed regions are labeled with the corresponding enlarged view of the inset on the right. These insets represent the multiple events of SNX27–retromer on MT1-MMP endosomal subdomains (N = 2, n = 6; scale bars = 5 µm, inset = 2 µm). (B) Diagrammatic representation of SNX27WT and its different deletion mutants used in the study. (C) MDA-MB-231 cells transfected with control siRNA or siRNA against SNX27 for 48 h and transfected again with siRNA-resistant GFP-fused SNX27WT and mutants. Cells were seeded on labeled gelatin and were fixed and stained with DAPI before the effect of gene suppression diminished. The degradation index was calculated as described earlier. Arrows represent the degradation dots. (N = 4, n = 200; scale bar = 10 µm). One-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The graph represents means ± SEM. (D) GFP-SNX27WT and GFP-SNX27Δ67–77 mutants were cotransfected along with mCherry–MT1-MMP. 12 h after transfection, cells were analyzed with a TIRF microscope. The number of MT1-MMP endosomes was counted (N = 3, n = 45; scale bar = 10 µm). Values in the graph represent mean ± SEM. Two-tailed Student’s t test, *** P < 0.001. N, number of experimental repeats.
Figure 5.

SNX27 and retromer reside on the endosomal subdomains of MT1-MMP to facilitate its recycling and matrix degradation activity. (A) MDA-MB-231 cells expressing GFP–MT1-MMP were fixed, immunostained with an antibody against SNX27 and Vps26, and analyzed by super-resolution microscopy 3D-SIM. Boxed regions are labeled with the corresponding enlarged view of the inset on the right. These insets represent the multiple events of SNX27–retromer on MT1-MMP endosomal subdomains (N = 2, n = 6; scale bars = 5 µm, inset = 2 µm). (B) Diagrammatic representation of SNX27WT and its different deletion mutants used in the study. (C) MDA-MB-231 cells transfected with control siRNA or siRNA against SNX27 for 48 h and transfected again with siRNA-resistant GFP-fused SNX27WT and mutants. Cells were seeded on labeled gelatin and were fixed and stained with DAPI before the effect of gene suppression diminished. The degradation index was calculated as described earlier. Arrows represent the degradation dots. (N = 4, n = 200; scale bar = 10 µm). One-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The graph represents means ± SEM. (D) GFP-SNX27WT and GFP-SNX27Δ67–77 mutants were cotransfected along with mCherry–MT1-MMP. 12 h after transfection, cells were analyzed with a TIRF microscope. The number of MT1-MMP endosomes was counted (N = 3, n = 45; scale bar = 10 µm). Values in the graph represent mean ± SEM. Two-tailed Student’s t test, *** P < 0.001. N, number of experimental repeats.

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