FIG. 4.

Role of ATR in growth arrest of ts13 cells. ts13 cells were incubated with the indicated concentrations of caffeine (Caff) for 2 h and either maintained at the permissive temperature (33.5°C) or shifted to the restrictive temperature (39.5°C) for an additional 24 h. Cells were fixed and stained for DNA content with propidium iodide. (A) Graph showing the percentage of cells in S phase in the presence of the indicated concentrations of caffeine. (B) FACS profiles of DNA content of caffeine-treated ts13 cells at the restrictive temperature for 24 h. (C and D) Cells were pretreated with caffeine for 2 h at 33.5°C, shifted to 39.5°C, and then cultured for the indicated for the indicated periods of time. Lysates were prepared and blotted with the indicated antibodies. (E) ts13 cells were cotransfected with WT or KD derivatives of ATR or ATM and GFP, which served as a marker of transfected cells. Cells were incubated at 33.5°C for 24 h and then either maintained at 33.5°C or shifted to 39.5°C for an additional 24 h. The graph shows the percentage of GFP-positive (transfected) cells in S phase. (F) FACS profiles of GFP-positive (transfected) and GFP-negative (nontransfected) cells cultured at the restrictive temperature for 24 h. Cyc, cyclin; Vin, vinculin.