FOCUS, SNU475 (1000 cell/well in 150 μL/well); Huh7, MCF7 (2500
cell/well in 150 μL/well); HepG2, Hep3B, SKBR3 (3000 cell/well
in 150 μL/well); PLC-PRF-5, Hep40 (5000 cell/well in 150 μL/well);
MDA-MB-231, MDA-MB-468, MCF10A (6000 cell/well in 150 μL/well);
and ZR-75 (7500 cell/well in 150 μL/well) were cultured in 96-well
plates and were inoculated in an incubator for 24 hours. The compounds
were dissolved in dimethyl sulfoxide (DMSO) (Sigma, St Louis, MO)
as a 20 mM stock solution. The treatment of cells with the compounds
was done in a concentration range starting from 40 to 2.5 μM.
The compounds which were below 2.5 μM were tested in a concentration
range from 2.5 to 0.15 μM. End of the 72h compound treatment,
the cells were fixed using 10% (v/v) trichloroacetic acid (MERCK)
for an hour and washed with ddH2O, and left for air-drying.
The fixed plates were stained with 50 μL of sulforhodamine B
(SRB) solution (Sigma) at RT for 10 min. To remove unbound SRB dye,
acetic acid was used to wash cells three times and left for drying.
The protein-bound SRB was solubilized with 10 mM Tris-base (Sigma)
and their absorbance was measured with a 96-well plate reader at 515
nm wavelength (ELx800, Biotek). The IC50 values of compounds
were calculated in comparison with control group DMSO. Data with R2 values larger than 0.9 are considered significant.