Immunofluorescence staining was performed in cells cultured on glass coverslips as described previously (25 (link),40 (link)). After fixation with 2% paraformaldehyde for 30 min at room temperature, the cells were incubated with anti-ASC (1:100; cat. no. sc-514414; mouse), anti-caspase-1 (1:100; cat. no. sc-1218-R; rabbit), anti-E-cadherin (1:200; cat. no. sc-21791; mouse) or anti-NF-κB (1:200; cat. no. sc-8008; mouse) antibodies (all Santa Cruz Biotechnology, Inc.) at 4°C overnight. After washing, the slides were incubated with Alexa Fluor 488-labeled goat anti-rabbit (1:500; cat. no. ab150077; Abcam) or Alexa Fluor 555-labeled anti-mouse secondary antibody (1:500; cat. no. ab150113; Abcam) for 1 h at room temperature and then subjected to examination using a Fluoview FV1000 confocal laser scanning microscope (Olympus Corporation). Images were analyzed and the co-localization coefficients were calculated using Image-Pro® Plus (version 7.01; Media Cybernetics, Inc.) (43 (link)). Caspase-1 was detected using Alexa Fluor-488 anti-rabbit IgG secondary antibody and ASC, NF-κB or E-cadherin using Alexa Fluor-555 anti-mouse secondary antibody. For the staining of NF-κB, nuclei were counterstained using Nuclear Green DCS1 (cat. no. ab138905; Abcam).