Growth Inhibition IC50 Assay.

Cells were seeded at various numbers, ranging from 800 to 8,000 per 80 µL, into 96-well plates according to cell growth rate 24 h before compound overlay and cultured at 37 °C and 5% CO₂. Compounds were prepared as 10-mM stocks in 100% DMSO. Each 10-mM stock was diluted with DMEM (Invitrogen) containing 10% (vol/vol) FBS such that the final concentrations ranged from 100 pM to 50 μM. Aliquots (20 μL) from each concentration were overlaid with the aid of a Tecan Freedom EVO robotic workstation (Tecan) to 80 μL of preseeded cells to achieve final concentrations of 20 pM to 10 μM. After 5 d, cell growth in each well was assessed by an SRB assay (described earlier). SRB absorbance values were adjusted by subtracting the average of the baseline readings from untreated cells assessed 1 d after cell seeding. Relative cell growth was calculated by comparing vs. DMSO-treated cells. The concentrations at which cell growth was inhibited by 50% (i.e., growth inhibition IC₅₀) were calculated by using GraphPad Prism software (GraphPad).