FIGURE 5:

HS has a gradual effect on DNA replication. (A) Human mcf-7 cells were pulse-labeled with BrdU (100 μM, 20 min), heat stressed (45.5°C, 30 min), and double-immunostained against γH2AX (red) and BrdU (green). Representative images are shown. Scale bar: 5 μm. (B) Human mcf-7 cells were pulse-labeled with BrdU (100 μM, 20 min), treated with aphidicolin (6 mM, 60 min), and double-immunostained against γH2AX (red) and BrdU (green). Scale bar: 20 μm. (C) A DNA fiber analysis (molecular combing) of the DNA replication speed under HS conditions. Human mcf-7 cells were heat-stressed for 30 min at different temperatures (42, 44, and 45.5°C) and allowed to recover at 37°C for 0, 0.5, 3, 6, and 24 h. After these time intervals, the cells were pulse-labeled with BrdU (100 μM, 20 min) and subjected to DNA fiber analysis. The tracks of incorporated BrdU were revealed by immunostaining. For each time point, the lengths of at least 150 tracks were measured, and the average replication velocity was calculated. The error bars represent the SEM. Representative images of the BrdU tracks are shown to the left of the graphs.