Cell culture and reagents

Time course cell viability assay

U2OS, B16-F10, or C33A cells purchased from the American Type Culture Collection were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Life Technologies, NY, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, MO, USA) and 1% penicillin-streptomycin (Gibco, Life Technologies, NY, USA) at 37°C under 5% CO2.

1^st day: A total of 5 × 10³ cells (100 μl) were seeded per well in five 96-well plates for the time course assays with 5 time points. 

2. 2^nd day: 24 hour after the seeding, the cells in the plates were synchronized with 1-hour dex (100 nM) pulse (100 μl/well) and were treated with vehicle (e.g., DMSO; n=6 wells) or anticancer drugs (0.01 μM, 0.1 μM, 1 μM; n=6 wells for each drug conc.) at 6-hour intervals over the course of 24 hours (24 hr, 30 hr, 36 hr, 42 hr, 48 hr) (Please see Fig. 2A).

4^th day: 48 hours later for each sample, time course of cell viability was determined colorimetrically with Alamar Blue reagent (DAL1025, Thermo Fisher Scientific) at 6 hour intervals (Please see Fig. 2A). Briefly, microplates were subjected to removal of media by aspiration followed by addition of a mixture of 100 μl of fresh media and 10 μl of Alamar Blue reagent. The plates were incubated for 6 hours at 37°C, and fluorescence activity was monitored at 555-nm excitation wavelength and 595-nm emission wavelength on the Epoch Microplate Spectrophotometer.