2500 cells per well were plated in 12-well plates (Greiner Bio One Cellstar, Frickenhauser - Germany) and were allowed to grow for about 4 to 5 days until small colonies could be clearly seen. Cells were treated for 48 hrs with different concentrations (2–100 nM) of staurosporine or UCN-01 (7-hydroxystaurosporine) in growth media. For each concentration datapoint of the two drugs, cells were analyzed in quadruplicates. Staurosporine was purchased as 1 mM ready-made solution in DMSO (Sigma Cat # S6942) and UCN-01 as powder (Sigma Cat # U6508). UCN-01 was diluted in DMSO according to the manufacturer's instructions. Cell culture plates containing colonies were gently washed with PBS and fixed with 3.7% formaldehyde for 10 minutes. Wells were rinsed once again with PBS and colonies were stained with 0.2% crystal violet solution in 10% ethanol for 10 minutes. Excess stain was removed by washing repeatedly with PBS. All the procedures were done at room temperature. The plates can be stored at room temperature or at +4 °C for several months without any visible fading of the dye.
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