Lysozyme solution

Coupon biofilms samples were washed with PBS (Thermo Fisher Scientific, MA, USA), fixed in 4% paraformaldehyde (Sigma Aldrich, UK) for 2 h at 4°C, washed again with PBS and 1 mL of dehydration buffer (50% ethanol in PBS), and placed at −20°C for 2 h. Subsequently, the samples were washed with PBS and incubated in 1 mL lysozyme solution (1 mg mL⁻¹) (Thermo Fisher, USA) for 15 min at 37°C. After another wash with PBS, hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl pH 7.2, 0.01% vol/vol SDS, and 25% vol/vol formamide) (Sigma Aldrich, UK) and 250 ng of the appropriate DNA probe (see Table 4) were added. The biofilms were incubated in the dark covered in aluminum foil for 3 h at 46°C. After incubation, the biofilms were washed with a wash buffer (10 mM Tris-HCl pH 9.0 and 1 mM EDTA) (Sigma Aldrich, UK) and incubated in this buffer at 55°C for 10 min. This step was repeated three times. All biofilms were kept in the dark until imaged with the Zeiss LSM880 confocal laser scanning microscope (Zeiss, Germany). Excitation intensity and any post-processing using the Zen Black software (Zeiss, Germany) were recorded within the software for accurate depiction of signal received.

The cells or tumor tissues were split using lysozyme solution (90,082, ThermoFisher, Shanghai, China), and the protein concentration of the cells was measured using a BCA kit (Solarbio, Beijing, China). Then, the protein samples were transferred to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis (Mini-Protean-3, Bio-Rad, Hercules, CA, USA) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Massachusetts, USA). After the membranes were blocked with 5% skim milk, they were incubated with primary rabbit anti-human antibodies against β-catenin (1:2000, ab16051, Abcam), CyclinD1 (1:200, ab16663, Abcam), GSK-3β (1:5000, ab32391, Abcam), p-GSK-3β (1:1000, ab131097, Abcam), and β-actin(1:2500, ab8227, Abcam, UK) and mouse anti-human FUT4 (1:1000, sc-19,648, Santa Cruz Biotechnology, Beijing, China). After three washes with TBST (TBS, 1 ml/L Tween-20), the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:2000, ab6721, Abcam) and goat anti-mouse immunoglobulin G secondary antibodies (1:2000, sc-2354, Santa Cruz Biotechnology). Finally, the enhanced chemiluminescence (ECL) method was used for detecting signals, and greyscale scanning and quantification of the protein bands were performed by ImageJ (NIH) software 6.0. The expression levels of proteins were normalized to β-actin.