Figure 3.

Effect of SB202190 (p38 inhibitor, 10 μM), UCN-01 (Chk1 inhibitor, 500 nM), or caffeine (ATM, ATR inhibitor, 2 mM) on G₂ checkpoint activation and G₂ exit. (A) After preincubation with DMSO or inhibitors for 30 min, the medium was changed: NaCl was added to total osmolality of 550 milliosmol/kg, as indicated. Percent of cells in mitosis was determined as in Fig. 1. (B and C) Cells were synchronized in early S by incubation for 11 h with 1 μM of the DNA polymerase inhibitor aphidicolin. Then the media were changed to media free of aphidicolin and made hypertonic by addition of NaCl to a total osmolality of 550 milliosmol/kg, as indicated. After 4 h, when G₂ arrest was complete at 550 milliosmol/kg, inhibitors or DMSO were added together with nocodazole (a microtubule inhibitor, 0.5 μM) to trap all cells that entered mitosis. (B) Representative cytograms showing the position of cells in the cell cycle at the time inhibitors were added (4 h). (C) Percent of cells in mitosis, based on P-H3 staining at the indicated time points.