Fig. 1.

Survivin knockdown induces DNA breaks and DNA damage response in breast cancer cell lines. DNA damage was evaluated in breast cancer cells 48 h after Survivin depletion using siRNA by γH2AX detection by immunoblot (a) and immunocytochemistry (b) and by single cell comet assay (c). a γH2AX and Survivin immunoblot analysis of Cal51 cells (left panel) treated by Cisplatin 6 mM (as positive control, lane 2) or not (untreated, lane 1), and transfected with siRNA control (siControl) (lane 3) or siSurvivin (lane 4). MDAMB-231 cells (middle panel) and MCF7 cells (right panel) depleted in Survivin (siSurv) or not (siCt). γH2AX staining by ICC (b) or single cell gel electrophoresis in alkaline buffer comet assay (c) were performed in 48 h Survivin-depleted or SiCt MDAMB-231 cells. 2 Gy-irradiated cells (b and c) were used as positive control. Representative images are shown (n = 5). Corresponding quantitative comet parameters were calculated using the comet assay software and comet tail moment was shown in c (below panel). d GFP nuclear foci were assessed in GFP-53BP1 expressing Cal51 cells 48 h after Survivin depletion or control. Representative images are shown in the upper panel. % of positive cells based on GFP nuclear foci were counted in each condition. Cisplatin treatment was used as positive control