N-cadherin is a key interactor of ARF3 that controls morphogenesis. (A and B) PC3 cells in 3D invasion assay. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/4. Scale bars, 100 μm. Magnified images of boxed regions shown. White and black arrowheads, invasive chain or sheet, respectively. RWD at t = Max1/4, normalized to Scr is shown in graphs (B). Data is mean ± SEM (5 experimental replicates, triangles, 3–8 technical replicates, circles. P values (Student’s two-tailed t test), **P ≤ 0.01 and ***P ≤ 0.001. (C) Phase images of PC3 acini expressing ARF3-mNG and Scr or N-cadherin shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 3 experimental replicates each with 3 technical replicates/condition. 4,039 (Scr), 4,814 (N-cadherin KD1), and 3,454 (N-cadherin KD2) mNG acini quantified in total. (D) Quantitation of C. Heatmaps, Area is mean of Z-score normalized values (purple to yellow). P values, Student’s t test, Bonferroni adjustment, represented by size of bubble. Heatmaps, Round, Spindle, or Spread is Log2 fold change from control (Scr; blue to red). Proportion of control at each time is Z-score normalized (white to black). P values, CMH test, Bonferroni adjusted, represented by size of bubble. Dot indicates P value (Breslow–Day test, Bonferroni-adjusted) for consistent effect magnitude. (E) Model, ARF3-RAB11FIP4 complex interacts with and regulates levels of N-cadherin in intracellular vesicles to control different modes of invasion.
