8 chamber glass slides

HUVECs were cultured to subconfluence on 8 chamber glass slides (BD Falcon, Bedford, MA). After treatment, they were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and permeabilized with 0.1% Triton X-100. Actin cytoskeleton was stained with Phalloidin-FITC. Cells were incubated with 1:400 diluted polyclonal anti-PAK4 antibody, followed by a sequential incubation with a 1:1000 diluted Alexa 488 or 568-conjugated secondary antibody. Nuclei were counter stained with DAPI (Sigma-Aldrich). The cells were imaged using a ZEISS 780 inverted confocal laser scanning microscope (ZEISS, Cambridge, UK) and analyzed by using Zen software.

PSN-1, HT1080, or HT1080/hCLDN4 cells (70,000 cells per well) were seeded onto 8-chamber glass slides (Falcon) and allowed to adhere overnight. Cells or frozen tumor xenograft sections (10 μm) were washed in phosphate-buffered saline and fixed using 4% paraformaldehyde solution (10 min). After washing, the sections were permeabilized using 0.5% digitonin for 15 min at room temperature, washed again, and blocked in 2% bovine serum albumen for 1 h at room temperature. The cells or sections were then exposed to anticlaudin-4 (PA5–28830; 1:100) or anticlaudin-3 (341700; 1:100) antibody at 4°C overnight, washed in phosphate-buffered saline (3 × 5 min), and incubated with goat antirabbit IgG Alexa Fluor 488 (1:500) for 1 h at room temperature. After further washing (3 × 5 min), the slides were mounted using Vectashield with 4′,6-diamidino-2-phenylindole (Vector H-1200). Fluorescence micrographs were acquired using a TCS SP8 confocal microscope (Leica Microsystems).