Figure 2.

Involvement of ATR, but not ATM, in PCC. (A) Induction of ATR-kd expression by doxycycline promoted PCC in U2OS cells treated with caffeine (1 mM) and/or hydroxyurea (HU, 1 mM). (B) ATR-wt expression diminished PCC caused by the combination of caffeine and hydroxyurea. (C) 293T cells were transiently transfected (see Materials and Methods), and expression of CMV promoter-driven constructs of ATR or ATM were compared by Western blot to endogenous levels present in control (green fluorescent protein-transfected) cells. (D) Transient transfection in 293T cells of ATR-kd synergized with low-level UV (200 J/m²) to cause PCC, but ATM expression had no effect. (E) Neither ATM-wt nor ATM-kd transfection had any effect on PCC induced by caffeine and hydroxyurea, whereas transfection of the ATR-wt and ATR-kd constructs into 293T cells had effects similar to those shown in A and B in which their expression had been induced in the U2OS cell lines by doxycycline. (F) Analogously, neither ATM-wt nor ATM-kd transfection had any effect on PCC induced by ATR-kd and UV, whereas additional ATR-kd augmented and ATR-wt diminished PCC. Cells were transfected with 2 μg of DNA (1 μg of ATR-kd and 1 μg of the indicated construct) and were treated with UV (200 J/m²) and nocodazole (10 ng/ml) 48 h later, then harvested for mitotic spreads 24 h later (see Materials and Methods).