The ATM inhibitors KU-55933 (S1092) and CP-466722 (S2245) were obtained from Selleckchem. LOPAC-1280 library, Nutlin-3 and Etoposide were purchased from Sigma-Aldrich.
Screening of the LOPAC small compound library was performed as follows: 10 000 cells per well were seeded in 96-well flat bottom plates (Corning CellBind CLS3340-50EA), 24 hours later cells were treated with drugs for 24 hours at a final concentration of 5μM in 0.5% DMSO in the presence of 0.5μM Etoposide, or with 0.5μM Etoposide in drug-free DMSO (negative control) or with DMSO only. Cells were then fixed for 10 minutes in 10% neutral buffered formalin solution (Sigma HT5012), nuclei stained for 10 minutes with DAPI (1μg/ml in PBS) and stored at 4°C until scanned (Thermo Cellomics Compartmental Analysis V4). Acquisitions were made with a 20x objective, both with XF93-Hoechst filter, 0.05sec exposure (DAPI) and XF93-TRITC filter, 0.5sec (RFP).
Screening of the LOPAC small compound library was performed as follows: 10 000 cells per well were seeded in 96-well flat bottom plates (Corning CellBind CLS3340-50EA), 24 hours later cells were treated with drugs for 24 hours at a final concentration of 5μM in 0.5% DMSO in the presence of 0.5μM Etoposide, or with 0.5μM Etoposide in drug-free DMSO (negative control) or with DMSO only. Cells were then fixed for 10 minutes in 10% neutral buffered formalin solution (Sigma HT5012), nuclei stained for 10 minutes with DAPI (1μg/ml in PBS) and stored at 4°C until scanned (Thermo Cellomics Compartmental Analysis V4). Acquisitions were made with a 20x objective, both with XF93-Hoechst filter, 0.05sec exposure (DAPI) and XF93-TRITC filter, 0.5sec (RFP).