Caspases 3/7 Activities Assay

Snb19 and LN229 cells were seeded on 96 well-plates at the initial density of 1 × 10⁴ cells/well with appropriate medium. After culturing for 24 h, cells were treated with TMZ and the top compound at IC₅₀ concentration for 5 h. Determination of caspase activity was performed using Caspase-Glo 3/7 Assay kit (Promega, Madison United States) followed by the standard protocol from the manufacture. Briefly, the plate containing cells were removed from incubator and allowed to equilibrate to room temperature for 30 min. An amount of 100 μl of Caspase-Glo reagent was added to the plate containing 100 μl of treated cell, untreated cell, blank or TMZ. After that, content of wells was gently mixed using a plate shaker at 300–500 rpm for 30 s. The plate was incubated for further 1 h before measuring the luminescence using a plate-reading luminometer (Fluoroskan Ascent FL, Thermo Labsystems). The fold increase in caspase 3/7 was calculated using formula (2) as described in ROS assay.