Cytotoxicity assay
In a 96 well plate, 5,000 U2OS WT cells were seeded and cultured for 24 hours under standard culture conditions. Treated cells with various concentrations of doxorubicin hydrochloride for 24 and 48 hours. After 24 and 48 h, LDH activity was measured using reagents from CyQUANT LDH Cytotoxicity Assay Kit (C20300, invitrogen). Absorbance was read at 490 nm and 680 nm to determine LDH activity. Percent cytotoxicity was calculated using the following: [(Compound-treated LDH activity-Spontaneous LDH activity)/(Maximum LDH activity-Spontaneous LDH activity)] *100.