Protrudin promotes cell invasion. The cell lines indicated were allowed to invade into plugs of fibronectin-supplemented Matrigel or collagen I for 4 d, stained with Calcein, and imaged by confocal microscopy. Optical sections (Δz = 10 µm) are shown for each condition. Graphs show the percentage of cell invasion over 40 µm. Each plotted point represents the average invasion of one plug. (A) In total n = 9 plugs (five z-stacks per plug) were analyzed from three independent experiments. Values represent mean ± SD. ***, P < 0.001, one-way ANOVA, Dunnett’s post hoc test. (B) In total, n = 18 plugs (five z-stacks per plug) were analyzed from six independent experiments. Values represent mean ± SD. ***, P < 0.001, one-sample t test. (C) In total, n = 12 plugs (five z-stacks per plug) were analyzed from four independent experiments. Values represent mean ± SD. *, P < 0.05, one-sample t test. (D) In total n = 9 plugs (5 z-stacks per plug) were analyzed from 3 independent experiments. Values represent mean ± SD. *, P < 0.05, one-sample t test. (E) Single tumor spheroids from RPE-1 and RPE-1-Protrudin–overexpressing cells were embedded in Matrigel and allowed to invade. Phase contrast micrographs show spheroids from days 0 and 2. Graphs represent percent increase of spheroid area and distance of the longest protrusion from the outer rim of the spheroid dense core. Values represent mean ± SD. Each plotted point represents one spheroid. In total, n = 34 spheroids from six different experiments. ***, P < 0.001, Mann–Whitney. OE, overexpression.