L3000

In total, 414 compounds with anti-cancer properties (Selleckchem L3000) were screened. HAP1-ABE^dox:GFP reporter cells were seeded at a density of 2 × 10⁴ per well in a 96-well Bio-One 655090 microplate (Greiner) with 0.5 μg/ml doxycycline. After 1 h of incubation, 10 μl of the test compound was added to each well using a Janus liquid handler (PerkinElmer) to generate final concentrations of 100 and 500 nM. Since the potency of each compound varied, we chose two concentrations (100 and 500 nM) for the initial screening as per the manufacturer's instructions and other studies (17 (link),18 (link)), and the concentrations of some candidate drugs was further optimized. The plates were incubated for 48 h and the cells were stained with Hoechst33342 dye. GFP expression levels were measured by capturing and analyzing the cell images with the Operetta High Contents Screening system (PerkinElmer). The hit compounds were selected according to the fold-change in GFP expression. Drug screens were performed with appropriate multiple controls (eight wells for dimethylsulfoxide [DMSO] as a negative control and eight wells for doxycycline as positive controls per plate). The mean of the Z-prime factor was 0.52 in the 100 nM screen and 0.09 in the 500 nM screen. The highest DMSO concentration in the screening was no more than 0.5%. All compounds were assessed using biological replicates.