Functional Assays for Head and Neck Cancer Cells

The procedures for conducting functional assays (cell proliferation, migration, and invasion assays) with HNSCC cells were described in earlier publications [23 (link),24 (link),25 (link),26 (link)]. In brief, for proliferation assays, Sa3 or SAS cells were transferred to 96-well plates at 3.0 × 10³ cells per well. Cell proliferation was evaluated using the XTT assay kit II (Sigma–Aldrich, St. Louis, MO, USA) 72 h after the transfection procedure. For migration and invasion assays, Sa3 and SAS cells were transfected in 6-well plates at 2.0 × 10⁵ cells per well. After 48 h, transfected Sa3 and SAS cells were added into each chamber at 1.0 × 10⁵ per well. Corning BioCoat^TM cell culture chambers (Corning, Corning, NY, USA) were used for migration assays whereas Corning BioCoat Matrigel Invasion Chambers were used for invasion assays. After 48 h, the cells on the lower surface of chamber membranes were stained and counted for analysis. All experiments were performed in triplicate.