FIGURE 3.

SFKs promote down-regulation of ATR-mediated Chk1 phosphorylation during G₂ checkpoint recovery. A, HeLa S3 cells were exposed to 110 nm Adriamycin (ADR) in G₂ phase as described under “Experimental Procedures.” Cells were allowed to recover for the indicated times, and SDS lysates were prepared for Western blotting. B, C, and D, cells were treated as in A except that 5 μm SU6656 was added 1 h before Adriamycin exposure. -Fold increases in band intensity induced by SU6656 are shown for samples with 11 h of recovery. Results are expressed as means ± S.D. E, instead of SU6656, 20 μm PP2 was used. F, HeLa S3 cells were treated as in B. One hour before harvest, 5 mm caffeine was added. G, H, and I, the same experiment as in B–E. J, HeLa S3 cells were synchronized by thymidine block for 16 h. Cells were exposed to 110 nm Adriamycin for 1 h from 6 h after release from thymidine block. At 11 h after the end of Adriamycin exposure, cells were harvested, and SDS lysates were prepared. For the indicated times before harvesting, cells were treated with 5 μm SU6656. The graph represents results obtained from two independent experiments. K and L, HeLa S3 cells were synchronized by double thymidine block. From 6 h after release from thymidine block, cells were exposed to 110 nm Adriamycin for 1 h. Cells were allowed to recover for the indicated times, and SDS lysates were prepared. Representative data from two independent experiments are shown. p values were calculated using t test. Error bars represent S.D. Async., asynchronously growing cells.