Cell Viability Assays of STK899704

Cell viability was quantified using a cell counting kit-8 (CCK-8) assay (Promega, Kumamoto, Japan). Approximately 0.7 × 10⁴ cells were seeded in each well of a 96-well plate containing 100 μL RPMI medium supplemented with 10% FBS, 100 μg/mL penicillin, and 0.25 μg/mL streptomycin and were cultured overnight. After 20 h of incubation, various concentrations of STK899704 were added to the wells and the cells were incubated further for the indicated time. Cell viability was analyzed by performing the CCK-8 assay according to the manufacturer’s instructions. Optical density was measured at 450 nm using a microplate reader (Apollo LB 9110; Berthold Technologies GmbH, Bad Wildbad, Germany). Also, cell viability was assessed by the MTS dye reduction assay, which measures mitochondrial respiratory function. Lung cancer cells were seeded (7 × 10⁴cells/mL) in 100 μL medium/well in 96-well plates, incubated overnight, and treated with various concentrations of STK899704, as described in the figure legends. Cell viability was calculated by assessing MTS metabolism as previously reported (Kwon et al., 2016 (link)). In brief, media samples (100 μL) were removed and incubated with 100 μL of MTS-PMS mix solution for 1 h at 37°C. Optical absorbance was measured at 492 nm using an ELISA reader (Apollo LB 9110, Berthold Technologies GmbH).