Determination of the IC50 values (i.e., the concentration of drug required to give half-maximal inhibition), cell cycle and apoptosis analysis

Triplicate cultures of the CB7, HEL, K562, WM9, MDA-MB-237 and PC3 cell lines were incubated with different concentrations of C54 or DMSO as a control for three days. Cell were subsequently subjected to an MTT assay by adding 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to the culture for 4 h. Following removal of the supernatant, 200 ml DMSO was added to dissolve the formazan crystals. The absorbance was read using a Synergy 2 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at 490 nm. For apoptosis and cell cycle analysis, CB7, HEL and K562 cell lines were incubated at 37°C with C54 or DMSO as a control for 24 h; subsequently, the cells were washed in cold phosphate-buffered saline (PBS). For the apoptosis experiments, cells were stained using an annexin V and propidium iodide (PI) Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA), prior to flow cytometric analysis. For cell cycle analysis, cells were fixed in cold 75% ethanol overnight at −20°C. Following a further wash with cold PBS, cells were stained in PI for 40 min at 37°C, and then subjected to flow cytometric analysis. IC₅₀ values were calculated using appropriate software.