Figure 3.

Effect of ssDNA oligonucleotides on the recruitment and activation of DNA repair proteins at DSBs in cells. (A) Intracellular localization of LNA ssO 4 h after lipofection in HCT116 cells. a and c panels correspond to mock-transfected cells and b and d panels to transfected cells. Red images correspond to Cy5 fluorescence from LNA ssO and gray images to light transmission acquisition. Cells were visualized with an Olympus IX73 inverted fluorescence microscope equipped with a DP26 digital camera and a 20× objective lens. (B) Characterization of the damage response induced by cell transfection with oligonucleotides. The LNA ssO alone or annealed with the complementary strand (dsO) (150 pmol each per well) was transfected for 2 h into HCT116 cells in 6-well plates. After cell lysis, proteins were separated on SDS-PAGE gel and electro-transferred on a membrane that was blotted with the indicated antibodies. (C) Pull-down experiment. HCT116 cells were transfected with a biotinylated ds DNA ± LNA ssO as indicated. ds probe and ssO amount per well were 4 and 100 pmol, respectively. After cell lysis, the extracts were incubated with streptavidin magnetic beads. Proteins bound to the beads or remaining in the soluble fraction were denatured and separated on SDS-PAGE gel; then electro-transfer on membrane and immuno-blotting (IB) with the indicated antibodies were performed. (D) Effect of oligonucleotides on the cellular response to DSBs. The LNA ssO (150 pmol per well) was transfected for 2 h into HCT116 cells in 6-well plates. After washing, cells were incubated for 30 min in culture medium containing 80 pM Calicheamicin-γ1. After cell lysis, proteins were separated on SDS-PAGE gel and electro-transferred on a membrane that was blotted with the indicated antibodies.