Fig. 1.

Optimizing activation and inhibition of ATR. A, U-2 OS cells were incubated with thymidine (2 mM) for 24 h and released for 3 h at which point nocodazole (100 ng/ml) was added for a further 12 h. Cells were released from nocodazole into fresh medium for the times indicated. Cells were fixed, stained with propidium iodide (PI) and analysed by FACS. B, U-2 OS cells synchronized in S-phase (11 h after release from nocodazole) were pre-incubated for 1 h with the indicated concentrations of berzosertib or gartisertib before addition of HU (1 mM) for 1 h. Cells were lysed, and extracts were subjected to Western blotting with the antibodies indicated. C, same as (B) except that S-phase cells were pre-incubated with berzosertib or gartisertib (1 μM for 1 h) before addition of HU (1 mM) for the times indicated. D, optimized cell treatment workflow.