GFP-Protrudin expression rescues cell invasion of Protrudin-KO cells, and the prognostic value of Protrudin in different cancer types. (A) The indicated cell lines were allowed to invade into plugs of fibronectin-supplemented Matrigel for 4 d, stained with Calcein, and imaged by confocal microscopy. Optical sections (Δz = 10 µm) are shown for each condition. Graphs show the relative cell invasion over 20 µm. Each plotted point represents the average invasion of one independent experiment, n = 4. In total, 12 plugs (five z-stacks per plug) were analyzed. Values represent mean ± SD. **, P < 0.01, one-sample t test. (B–D) Survival curves are plotted for breast, gastric, and ovarian cancer using the publicly available database Kaplan-Meier Plotter for high and low expression of Protrudin (ZFYVE27 Affymetrix ID 225281_at). (B) Analysis of lymph node–positive, estrogen receptor–negative, progesterone receptor–negative breast cancer patient cohorts (hazard ratio [HR], 1.88; 95% confidence interval [CI], 1.09–3.24. (C) Analysis of metastasis-free gastric cancer patient cohorts (HR, 1.62; 95% CI, 1.22–2.14). (D) Analysis of ovarian cancer patient cohorts (HR, 1.44; 95% CI, 1.19–1.76).