Effect of different treatments on factory number. (a) After synchronization in early S phase as described in Fig. 1, U2OS cells were irradiated with 1–5 Gy γ ray. 2 h after irradiation, cells were pulsed with Cy3-dUTP. Both the number and intensity of the Cy3-dUTP foci were measured in each cell. The mean and SEM were derived from >50 cells. (b) 2 h after irradiation with 1–5 Gy γ ray or 20 J/m2 UV in the presence or absence of 10 µM ATM inhibitor KU55933, total cell lysate was immunoblotted for phospho-CHK1, phospho-CHK2, and actin. (c) After synchronization in early S phase, U2OS cells were treated with 0–250 µM roscovitine for 2 h and pulse labeled with Cy3-dUTP. Both the number and intensity of Cy3-dUTP foci were measured in each cell. The mean and SEM were derived from >50 cells. (d and e) Cells were synchronized in early S phase and treated with HU or 20 J/m2 UV. 2 h after treatment, cells were lysed for immunoprecipitation with Cdk2 or cyclin A antibody. The associated kinase activity of the immunoprecipitates was assayed on histone H1.
