Cell viability assay

The inhibitory effects of PI-1840 on the OS cells were evaluated using CCK-8 assays. A cell suspension was produced from OS cells, and the cells were counted. Subsequently, the cells were seeded into a 96-well plate at a density of 5×10³ cells/well, and incubated for 12 h to allow attachment of the cells to take place. The cells were then treated with various concentrations of PI-1840 (0, 5, 10, 20, 40, 80 and 160 µM) for 24 or 48 h. CCK-8 solution was mixed with serum-free medium at a concentration of 10%, and subsequently replaced with the former medium without any bubbles during the procedure. The plate was then incubated for 1–4 h in the incubator at 37°C. Finally, the absorbance was measured at 450 nm using a microplate reader (BioTek China, Beijing, China).