FIG. 3.

DNA damage foci in ts13 cells at the restrictive temperature. (A) ts13 cells were cultured at the permissive temperature (33.5°C), shifted to the restrictive temperature (39.5°C) for 2 h, or incubated in 300 μM HU at 33.5°C for 2 h. Cells were immunostained with antibodies to ATR (1:200) or γ-H2AX (1:10,000) and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Images for ATR and γ-H2AX were merged. (B) ts13 and ts13R cells were cultured at 39.5°C for 2 h and immunostained with ATR and γ-H2AX antibodies and counterstained with DAPI. (C) ts13 cells were cultured at 39.5°C for 2 h and then briefly extracted with detergent prior to fixation and immunostaining with ATR and RPA34 (1:10,000) antibodies and counterstaining with DAPI. (D) ts13 cells were cultured at 39.5°C for 2 h or treated with IR (10 Gy) and incubated at the permissive temperature (33.5°C) for 90 min. Cells were fixed and stained with antibodies to P-ATM (1:100) and γ-H2AX and counterstained with DAPI.