In Vitro Cell Viability Assay

Cell viability was measured using an MTT assay. JN-DSRCT-1 cells were cultures until the log-phase and were subsequently seeded into a 96-well plate at a density of 10⁴ cells/well overnight to treatment with different concentrations of ONC201 (0.625-20 μM) or DMSO. Following an incubation of 72 h, the cells were then incubated with medium containing MTT for 4 h and the formazan crystals were dissolved with 150 μl DMSO. The plates were incubated on a shaker for 15 minutes at room temperature and the absorbance was measured at 490nm using a microplate reader (DTX880; Beckman Coulter). The cytotoxicity of the ONC201 was expressed either as percentage cell viability or as ratio of treated/DMSO. IC₅₀ values were calculated by sigmoidal dose-response curve fit using Prism GraphPad 6.0.