Xtt assay kit 2

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.

The tumor-suppressive functions of miRNAs were evaluated by transient transfection assays using mature miR-139-5p and miR-139-3p. The tumor-promoting functions of KRT80 (loss-of-function assays) were assessed by siRNA transfection assays using siRNAs targeting KRT80. Functional assays (proliferation, migration, and invasion assays) were performed according to procedures of previous studies [51 (link),52 (link)]. Briefly, for proliferation assays, HCT116 or DLD-1 cells were transferred into 96-well plates at 3.0 × 10³ cells/well. Cell proliferation was assessed using XTT assay kit II (Sigma-Aldrich, St. Louis, MO, USA) 72 h after the transfection procedure. For the migration and invasion assay, HCT116 and DLD-1 cells were transfected in 6-well plates at 3.0 × 10⁵ cells/well; 48 h later, transfected HCT116 and DLD-1 cells were added to each chamber at 1.0 × 10⁵ cells/well. Corning BioCoat^TM cell culture chambers (Corning, Corning, NY, USA) were used for the migration assay and Corning BioCoat Matrigel Invasion Chambers were used for the invasion assay. cells on the underside of the chamber membrane were stained and counted for analysis. All experiments were performed in triplicate. The details of the reagents used in these analyses are listed in Table S2.

Cells were seeded at 1.6 × 10⁵ cells/mL in 100μL of medium per well of a 96-well plate (Corning) for 24 h before treating with a range of concentrations (from 1 to 10⁴ nM) of E7107 and/or idasanutlin for 72 h. Concentrations are indicated in the figure legends. XTT Assay Kit II (Sigma-Aldrich, Gillingham, UK) was used to measure growth inhibition compared to solvent DMSO control.