Fig 1: ATR inhibition causes Cdc7-dependent MCM4 hyperphosphorylation in the chromatin fraction of S-phase cells. Cells were fractionated as described in Methods section. Western blotting of the nuclease-insoluble chromatin fraction is shown. a 293T cells were treated with 5 µM AZD6738, 10 µM Ku55933, or 5 µM ETP46464, 5 µM Ve-822, or 100 nM UCN01 for 1 h. b 293T cells were treated with 5 µM AZD6738 or 100 nM UCN01 for 1 h, the nuclease-insoluble fraction was treated with CIP or just incubated at 37 °C. c U2OS cells were synchronized by double thymidine-nocodazole block and then released into mitosis. G1 phase cells (6 h later) or S-phase cells (12 h later) were treated with 5 µM AZD6738, 5 µM ETP46464, 10 µM Ku55933, or DMSO for 1 h. d BJ-hTERT cells were synchronized by contact inhibition and then released by splitting (for S phase 10 h later) or kept confluent (for G1 phase). Cells were treated with 5 µM AZD6738, 5 µM Ve-822, 10 µM Ku55933, or DMSO for 1 h. e Cdk2 or Cdc7 inhibitor of 10 µM were added to 293T cells 15 min before 1 h treatment with 5 µM AZD6738. After fractionation, the nuclease-insoluble fraction was analyzed by western blotting. f Increasing concentrations (2, 5, 10 µM) of Cdk2 or Cdc7 inhibitor were added to 293T cells 15 min before 1 h treatment with 5 µM AZD6738. After the fractionation, the nuclease-insoluble fraction was analyzed by western blotting. g 293T cells were treated with 5 Gy IR, 10 J m-1 UV or 5 mM HU 30 min before the addition of 5 µM AZD6738 for another 30 min. h Picture indicates the conditions for the first 30 min of treatment and for the second 30' of treatment. After the second 30 min, treatment cells were fractionated and analyzed