Protrudin promotes degradation of extracellular gelatin in a dose-dependent fashion.(A) MDA-MB-231 cells were transfected with siRNA targeting Protrudin, FYCO1, RAB7, TKS5, or control siRNA. The cells were grown on coverslips coated with Oregon Green gelatin for 4 h and analyzed by confocal microscopy. Representative micrographs show degradation of fluorescent gelatin indicated by black areas. (B) Quantifications of images in A. The graph shows the relative area of gelatin degradation in the different knockdown conditions compared with siControl. Each individual data point represents the average of one independent experiment. Shown is mean ± SEM, n = 3 experiments (Protrudin, FYCO1#1, RAB7, and TKS5), n = 4 experiments FYCO1#2. **, P < 0.01; ***, P < 0.001, one-sample t test. Number of cells in total: siControl(Protrudin/TKS5), 1,145; siProtrudin#1, 800; siTKS5, 633; siControl(FYCO1#1), 1,064; siFYCO1#1, 975; siControl(FYCO1#2), 893; siFYCO1#2, 1,007; siControl(RAB7), 577; siRAB7, 512. (C) MDA-MB-231 and MDA-MB-231-GFP-Protrudin-sires#1 cells were transfected with siRNA targeting Protrudin (oligo #1 or #2) or control siRNA. 4 d after transfection, cells were grown on coverslips coated with Oregon Green gelatin for 4 h, stained with Rhodamine-Phalloidin (actin), and analyzed by confocal microscopy (knockdown verification in Fig. S1 A). Representative micrographs show degradation of fluorescent gelatin indicated by black areas. (D) Quantifications of images in C. The graph shows area of gelatin degradation in the different knockdown conditions compared with siControl. Note that the reduced gelatin degradation in Protrudin-depleted cells is rescued in the presence of GFP-Protrudin. Each individual data point represents the average of one independent experiment. Values represent mean ±SEM, n = 4. ***, P < 0.001, one-way ANOVA, Tukey’s post hoc test. In total, >700 cells were analyzed per condition. (E) RPE-1 cells or RPE-1-Protrudin-overexpressing cells were grown on coverslips coated with Oregon Green gelatin for 6 h with addition of HGF (50 µm) for the last 2 h, stained with Rhodamine-Phalloidin (actin), and analyzed by confocal microscopy. Representative micrographs show degradation of fluorescent gelatin indicated by black areas. (F) Quantification of images in E. The graph shows relative area of gelatin degradation. Each individual data point represents the average of one independent experiment. Values represent mean ± SEM, n = 5. **, P < 0.01, one sample t test. In total, >1,600 cells were analyzed per condition.
Figure 7.

Protrudin promotes degradation of extracellular gelatin in a dose-dependent fashion. (A) MDA-MB-231 cells were transfected with siRNA targeting Protrudin, FYCO1, RAB7, TKS5, or control siRNA. The cells were grown on coverslips coated with Oregon Green gelatin for 4 h and analyzed by confocal microscopy. Representative micrographs show degradation of fluorescent gelatin indicated by black areas. (B) Quantifications of images in A. The graph shows the relative area of gelatin degradation in the different knockdown conditions compared with siControl. Each individual data point represents the average of one independent experiment. Shown is mean ± SEM, n = 3 experiments (Protrudin, FYCO1#1, RAB7, and TKS5), n = 4 experiments FYCO1#2. **, P < 0.01; ***, P < 0.001, one-sample t test. Number of cells in total: siControl(Protrudin/TKS5), 1,145; siProtrudin#1, 800; siTKS5, 633; siControl(FYCO1#1), 1,064; siFYCO1#1, 975; siControl(FYCO1#2), 893; siFYCO1#2, 1,007; siControl(RAB7), 577; siRAB7, 512. (C) MDA-MB-231 and MDA-MB-231-GFP-Protrudin-sires#1 cells were transfected with siRNA targeting Protrudin (oligo #1 or #2) or control siRNA. 4 d after transfection, cells were grown on coverslips coated with Oregon Green gelatin for 4 h, stained with Rhodamine-Phalloidin (actin), and analyzed by confocal microscopy (knockdown verification in Fig. S1 A). Representative micrographs show degradation of fluorescent gelatin indicated by black areas. (D) Quantifications of images in C. The graph shows area of gelatin degradation in the different knockdown conditions compared with siControl. Note that the reduced gelatin degradation in Protrudin-depleted cells is rescued in the presence of GFP-Protrudin. Each individual data point represents the average of one independent experiment. Values represent mean ±SEM, n = 4. ***, P < 0.001, one-way ANOVA, Tukey’s post hoc test. In total, >700 cells were analyzed per condition. (E) RPE-1 cells or RPE-1-Protrudin-overexpressing cells were grown on coverslips coated with Oregon Green gelatin for 6 h with addition of HGF (50 µm) for the last 2 h, stained with Rhodamine-Phalloidin (actin), and analyzed by confocal microscopy. Representative micrographs show degradation of fluorescent gelatin indicated by black areas. (F) Quantification of images in E. The graph shows relative area of gelatin degradation. Each individual data point represents the average of one independent experiment. Values represent mean ± SEM, n = 5. **, P < 0.01, one sample t test. In total, >1,600 cells were analyzed per condition.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal