Glass bottom dish

TR146 cells were seeded in a glass-bottom dish (Corning) at a density of 5 × 104 cells/dish and incubated at 37 °C under 5% CO₂ for 24 h to allow the cells to adhere. The culture medium was discarded and replaced with fresh, serum-free medium containing serially diluted nanoliposomes (100 μg/mL, final concentration). The cells were incubated at 37 °C under 5% CO₂ for 2 h, washed three times with pre-chilled PBS at 4 °C, and fixed with 4% paraformaldehyde in PBS (w/v) for 20 min at room temperature (RT). The cells were rinsed three times with PBS and permeabilized with 0.5% Triton X-100 for 5 min at RT. The cells were then washed three times with PBS, stained with 500 μL TRITC phalloidin (Beyotime Biotechnology), and incubated for 50 min at RT. After washing three times with PBS, the cells were counterstained with 500 μL DAPI and stored at 4 °C. Fluorescence was observed under an SP8X confocal laser scanning microscope (Leica, Wetzlar, Germany). DAPI (green fluorescence) was measured at excitation and emission wavelengths of 364 and 454 nm. TRITC (red fluorescence) was measured at excitation and emission wavelengths of 545 and 570 nm. FITC-labeled insulin (green fluorescence) was measured at excitation and emission wavelengths of 490 and 525 nm.

SCC-15 and SCC-25 cells (5.0 × 10³ cells) were seeded on a glass-bottom dish (Corning, USA) and cultured with cisplatin (1 μg/mL) for 24, 48, and 72 h and with different concentrations of cisplatin (0, 1, 2, and 4 μg/mL) for 24 h. Regular media was used as the control. Primary antibodies to β-catenin (8480S, 1 : 100, Cell Signaling Technology) were added to the cells and incubated at 4°C overnight. After washing, FITC-conjugated goat anti-rabbit IgG (1 : 1000, Cell Signaling Technology) was added and incubated at room temperature in the dark for 2 h and then covered with Prolong® Gold Antifade Reagent and DAPI for 10 minutes at room temperature (#8961, 4,6-diamidino-2-phenylindole; Sigma). Confocal images were taken with a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss, Jena, Germany).