U tv camera

Migration and invasion assays were performed in 24-well cell culture plates with 8.0-µm-pore Transwell inserts (Corning, Inc., Corning, NY, USA). For the invasion assay, the membranes were coated with Matrigel (BD Biosciences, San Jose, CA, USA), and the subsequent steps were the same as those in the migration assay. HSC-2 cells (8 × 10³) and SCC-9 cells (4 × 10⁴) were transfected with siRNA-HIF-1α or siRNA-Par3 and cultured in inserts in Opti-MEM (Gibco) at 37 °C in the presence of 5% CO₂ overnight. The next day, Opti-MEM was added to the upper chamber and 10% FBS medium to the lower chamber, and the plates were incubated for 24 h. The inserts were fixed and stained with Mayer’s hematoxylin histological staining reagent (Dako, Santa Clara, CA, USA). After washing the inserts with PBS, migrated and invaded cells were counted under an inverted light microscope using an Olympus U-TV camera (Olympus, Tokyo, Japan). Migrated or invaded cells were counted in eight random fields. All assays were performed in triplicate.

Cell proliferation was assayed using the Click-iT™ 5-ethynyl-2′-deoxyuridine (EdU) Cell Proliferation Kit (Abcam, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, HSC-2 and SCC-9 cells at a density of 2 × 10⁵ were seeded on coverslips in 6-well plates and incubated with 10 µM EdU at 37 °C for 2 h. Then, the cells were washed with PBS, fixed with 4% PFA, and permeabilized with 0.5% Triton X-100 for 15 min. Following a brief wash with PBS-T, the click reaction cocktail provided in the Click-iT™ kit was added to the coverslips, which were then incubated in the dark at room temperature for 30 min. After washing with PBS-T, the samples were stained with DAPI for 10 min. Images were captured under a fluorescence microscope using an Olympus U-TV camera, and EdU-labeled cells were counted using the ImageJ software.