Figure 3.

8-oxoG excision is impaired in the absence of mediator or cohesin. (A) Relative 8-oxoG levels were determined by immunofluorescence using an antibody against the lesion before and at different times after exposure to KBrO₃ in control cells or cells depleted for SMC3 or MED14. NT = non-treated, K0H, K4H and K8H = immediately, 4 and 8 h after treatment, respectively. More than 1000 cells were analysed for each of the conditions and presented results correspond to a representative experiment out of three independent ones. The mean of 8-oxoG measured immediately after exposure to KBrO₃ (K0H) for each siRNA was set to 1 and used for normalization. Statistical analysis used a Kruskal–Wallis test. (****) P < 0.0001. (B) Representative images of 8-oxoG (green) detected by immunofluorescence in non-treated (NT) and KBrO₃ treated cells, at 0 (K0) or 3 (K3) hours after the treatment in control cells or cells depleted in MED14. A maximal projection of a z-stack (49 slices every 150 nm) is shown. The mask of the nucleus obtained using propidium iodide (PI) staining is shown. Higher magnifications for PI and 8-oxoG are shown in the right panels. A 3D projection of the 8oxoG foci through the entire nuclear volume is shown. The color scale illustrates the volume of the foci from smaller (white) to bigger (blue). Scale bar: 10 μm. (C) OGG1 activity in total cell extracts from cells depleted for MED14 or SMC3 compared to control cells. Cleavage of an 8oxoG-containing oligonucleotide by increasing amount of protein total extracts.