Atr inhibitor az20

Transcription inhibition was performed either by adding DRB (100 μM final concentration, Sigma-Aldrich), alpha-Amanitin (AMA, 20 μg/mL final concentration, Sigma-Aldrich) or Flavopiridol hydrochloride hydrate (FLV, 10 μM final concentration, Sigma-Aldrich) to the culture medium at 37°C 4 h before harvesting the cells. Inhibition of replicative synthesis was performed by adding Aphidicolin (Aphi, 1 μg/mL final concentration, Sigma-Aldrich) to the culture medium at 37°C 5 h before harvesting the cells. For ATR and ATM inhibition, cells were incubated with 2 μM ATR inhibitor AZ20 (Selleckchem) or 10 μM ATM inhibitor KU55933 (Tocris Bioscience) 1h before subsequent cell treatment. For FACT trapping, cells were incubated with 2 μM CBL0137 (Bertin Pharma) 15 min before subsequent cell treatment. The DSB-inducing agent neocarzinostatin (NCS, Sigma-Aldrich) was applied for 15 min at 50 ng/ml followed by 1 h recovery in fresh medium. For cell synchronization in late G2, cells were treated for 16 h with 2 mM Thymidine (Sigma-Aldrich), followed by 6 h release in fresh medium and 22 h treatment with the cdk1 inhibitor RO-3306 (10 μM final, Sigma-Aldrich).