(A) SiHa cells were incubated with the indicated concentrations of Ganetespib (HSP90-i) for 30 min at 37°C, then for an additional hour at 37 or 42°C. Next, cells were lysed and lysates were analyzed by western blotting, using antibodies against BRCA2 and Cyclin A (loading control). (B) Cells were treated as in (A), except Ganetespib was used at a concentration of 30 nM. At the indicated time after treatment, cells were irradiated with α-particles, fixed 30 min later and stained using antibodies against γH2AX (red) and RAD51 (green). The pictures are representative for cells irradiated 6 h after the treatment. The graphs show average percentage of cells containing tracks of γH2AX foci that were also positive for RAD51. (C) Cells were treated and analyzed as in (A), except the duration of incubation at elevated temperature was 0, 30 or 60 min. Equal sample loading was confirmed by probing for ORC2.