Figure 4

Analysis of the formation of reactive oxygen species (ROS) in irradiated Rec8-GFP expressing horsetail cells by di-hydroethidium (DHE, blue) staining.

ROS exposure converts DHE to oxidized ethidium (oxo-Eth, red). Image recording was normalized by addition of fluorescent TetraSpecks. (A) Control cell (0 Gy) with a Rec8-GFP tagged (green) nucleus showing blue reduced DHE accumulations in the cell. 200 Gy: X irradiated cell displaying oxidized DHE (ethidium, ox.DHE; red) in the entire cell, while the nucleus appears reddish due to ethidium binding to DNA. Cell 1 h post IR still showing slight red cytoplasmic labelling, while the nucleus appears orange due to colocalization of green Rec8-GFP and red Ethidium (ox.DHE). Still, non-oxidized blue DHE aggregates can be seen. H₂O₂: Cells treated with H₂O₂ showing cytoplasmic red ROS labelling and a green horsetail nucleus at 50 mM H₂O₂, while 100 mM H₂O₂ quenched any endogenous fluorescence except for the red ethidium (ox.DHE) label. The red dot represents an unknown ethidium-affine structure in the cytoplasm. For a split channel display of the images see Suppl. Fig. S2. (B) Irradiation induces persistent ROS in 90% of 200 Gy-irradiated cells. (C) H₂O₂-induced ROS – a dose of 100 mM renders values similar to 200 Gy X irradiation.