Figure 8.

Protrudin mediates cleavage of collagen-I. (A) MDA-MB-231 cells were embedded in fluorescent type I collagen (cyan, 2.0 mg/ml) for 24 h and stained for the cleaved collagen neo epitope using anti-Col-3/4C. The confocal micrograph shows the presence of collagen cleavage sites lining the collagen-I fibrils indicating the specificity of the antibody. (B) Protrudin KO decreases pericellular collagenolysis in MDA-MB-231 cells. MDA-MB-231, MDA-MB-231-Protrudin KO#1, and MDA-MB-231-Protrudin KO#1-GFP-Protrudin cells were embedded in fluorescent type I collagen (cyan, 2.0 mg/ml) for 24 h and stained with anti-Col-3/4C. Representative micrographs show the sum projections of confocal z-stacks from each condition. The graph shows quantification of the area of pericellular collagenolysis (µm²) from sum projections of wide field images. Each plotted point represent the average of one collagen droplet. n = 7 droplets (10 z-stacks per droplet) were analyzed from five different experiments. Values represent mean ± SEM. *, P < 0.05; ***, P < 0.001, one-way ANOVA, Tukey’s post hoc test. In total, >700 cells were analyzed per condition. (C) RPE-1 cells or RPE-1-Protrudin-overexpressing cells were embedded in fluorescent type I collagen (cyan, 2.0 mg/ml) for 24 h and stained with anti-Col-3/4C and Alexa488-Phalloidin (actin). Representative micrographs show the sum projections of confocal z-stacks from each condition. The graph shows quantification of the relative area of pericellular collagenolysis from sum projections of wide-field images. Each plotted point represents the average of one collagen droplet. n = 7 droplets (10 z-stacks per droplet) were analyzed from three different experiments. Values represent mean ± SEM. **, P < 0.01, one-sample t test. In total, >290 cells were analyzed per condition.