Mica microscope

Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (G4000, Promega, Madison, WI, USA) and the ATPlite assay (6016943, Perkin Elmer, Waltham, MA, USA). SW480^ctrl, SW480^KCTD1−, CACO^2ctrl, and CACO2^KCTD1+ cell lines were seeded in 96-well plates at a density of 8 × 103 cells per well. Absorbance was measured at 490 nm for MTT, while the ATPlite luminescence signal was detected using an automatic plate reader (Victor Nivo, Perkin Elmer, Waltham, MA, USA). MTT and ATPlite assays were conducted in triplicate, with similar results. For the cell migration assay, SW480^ctrl and SW480^KCTD1− cell lines were seeded into the upper Transwell chambers (BD Biosciences, San Jose, CA, USA). After incubation for 24 h, cells that had invaded through the chamber membrane were fixed with 11% glutaraldehyde solution (Sigma-Aldrich, St. Louis, MO, USA ) for 90 min, stained with crystal violet solution and, after elution, quantified through spectrophotometric measurement of optical density (O.D.) at 550 nm. The same experiment was conducted to visualize the invaded cells using a DAPI fluorophore and MICA microscope (Leica, Wetzlar, Germany).

Fifty thousand cells/well were seeded into a 6-well plate coated with polyHEMA and the obtained spheroids were collected, transferred into 6-well plates with coverslips on the bottom, and stimulated as above. At 3 and 48 h from stimulation, the cells were fixed with 4% formaldehyde (Carlo Erba Reagents, Emmendingen, Germany) and used in the immunofluorescence evaluation of E-cadherin and N-cadherin markers. Details were reported in the Supplementary Materials. Slides were visualized using a Leica MICA microscope in confocal mode.

S. flexneri 2a were grown as described above, and a 100 µL suspension (10⁷ CFU/mL) was added to microscope chamber slides. Bacteria were fixed in aqueous 4% paraformaldehyde for 45 min at RT and then washed with PBS. After washing, fixed bacteria were permeabilized and blocked for 1 h at with PBS containing 15% FBS, 2% BSA, and 0.1% Saponin (all from Sigma-Aldrich), then rinsed with PBS and incubated overnight at 4 °C with pooled sera from mice immunized IM with VirGα or AdjuPhos® diluted 1:100 in PBS containing 15% FBS and 0.2% BSA. Stained bacteria were washed twice with PBS and incubated with AF555-labeled goat anti-mouse IgG (Catalogue # A32727, Thermo Fisher Scientific) diluted 1:100 in PBS for 1 h at RT in the dark. After washing, bacteria were mounted in ProLong Gold antifade reagent with 4,6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology, Danvers, MA) for DNA staining at 4 °C for at least 24 h. Confocal images were obtained using a MICA microscope (Leica, Wetzlar Germany). Images were captured with a 60× water immersion objective, and settings were adjusted to optimize the signal. Images were collated using FIJI/ImageJ (NIH). Signal processing was applied equally across the entire image.