Prolong gold mount gel

T84 cells were grown on Transwell filters (pore size 0.4 μm) and treated with ASP in HBSS supplemented with 15 mM HEPES, pH 7.4. After incubation for 6 hr at 37°C in a 5% CO₂ atmosphere, the monolayer was washed three times in ice-cold phosphate-buffered saline (PBS) and fixed/permeabilized in ice-cold 100% ethanol at −20°C for 20 min. After being blocked with BlockAid Blocking Solution (Thermo Fisher Scientific, Waltham, MA), the samples were incubated with the primary antibodies against nectin-2 (ab135245, Abcam)1:250, afadin (A0349, Sigma-Aldrich)1:500, and E-cadherin (610181, BD Biosciences)1:500. The secondary antibody was Cy5-conjugated goat anti-rabbit antibody (ab6564, Abcam)1:1000 or FITC-conjugated goat anti-mouse antibody (sc-2010, Santa Cruz Biotechnology)1:200.
The samples were then washed three times with PBS and mounted in ProLong Gold mount gel (Thermo Fisher Scientific) with propidium iodide (PI; Nacalai Tesque). Fluorescence signals were visualized with a confocal laser scanning microscope (FV-300, Olympus, Tokyo). All experiments were performed in triplicate, and the reproducibility of the results was confirmed.

T84 cells were grown on Transwell filters (pore size 0.4 µm) and treated with various bacterial solutions suspended in HBSS containing 15 mM HEPES and pH 7.4 at MOI = 5. After incubation for 3 hr at 37°C in a 5% CO₂ atmosphere, the monolayer was washed three times in ice-cold PBS and fixed/permeabilized in ice-cold 100% ethanol at −20°C for 20 min. After being blocked with BlockAid™ Blocking Solution (Thermo Fisher Scientific), the samples were incubated with the primary antibodies against ZO-1 (610966, BD Biosciences) 1:500 and claudin-7 (ab27487, Abcam) 1:100. The secondary antibody was Cy5-conjugated goat anti-rabbit antibody (ab6564, Abcam) 1:1000 or Alexa Fluor 594-conjugated goat anti-mouse antibody (ab150116, Abcam) 1:1000. Samples were washed with PBS, and then 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) was applied for 10 min to stain the nuclei. After a final wash with PBS, the membranes were transferred to a glass slide and mounted in ProLong™ Gold mount gel (Thermo Fisher Scientific). Fluorescence signals were visualized with a confocal laser scanning microscope (LSM800, Carl Zeiss, Oberkochen, Germany).