Fig 2: OGG1 knockdown or inhibition sensitizes cancer cells to radiation(A) DLD-1 cells were transfected with three distinct siRNA against OGG1 or with a scramble RNA. OGG1 expression was monitored by RT-qPCR analysis and immunoblotting. (B) Cells were submitted to irradiation and their clonogenic efficiency was determined. Cloning efficiency of unexposed cells was set at 100%. Results of triplicate experiments are shown. Error bars represent standard error. ***p < 0.001; **p < 0.01.; *p < 0.05; Student's t-test. (C) Results from clonogenic efficiency data in Figure 7B are represented as line graphs, with all un-irradiated cells set at 100%. Results of triplicate experiments are shown. Error bars represent standard error. ***p < 0.001; **p < 0.01.; *p < 0.05; Student's t-test comparing each siRNA OGG1 against the scramble RNA control. 1: OGG1 siRNA 1; 2 : OGG1 siRNA 2; 3: OGG1 siRNA 3. (D) 8-oxoG cleavage assay was performed using 50 nM OGG1 and BSA in the presence of 10 μM. Chembridge 5552704 and 5245457 compounds or vehicle (DMSO). The two compounds inhibit the reaction. (E) DLD-1 colorectal cancer cells were irradiated (0 and 2 Gy) in the presence of 10 μM of Chembridge 5552704 and 5245457 compounds or vehicle (DMSO). Thymidine incorporation was measured for 3 days and is reported relative to the DMSO 0 h value.

Fig 3: CUT domains are sufficient for the recruitment to DNA damage sites(A) Diagrammatic representation of the constructs. The evolutionarily conserved domains of CUX1 are shown: CC, coiled-coil; C1, C2 and C3, CUT domains 1, 2 and 3; HD, homeodomain. NLS, nuclear localization signal. In the absence of the Cut homeodomain, a nuclear localization signal (NLS) is required to target C1C2 to the nucleus. (B) U2OS cells were transfected with plasmids expressing p200 CUX1-GFP, C1C2-NLS-GFP, OGG1-GFP, or Ku-mcherry fusion proteins. A 405 nm UV laser was used to induce DNA damage and the recruitment of GFP fusion proteins to the site of damage was followed in real-time. See Supplementary Movies 2–5.

Fig 4: Cut domains and OGG1 Accelerate Repair of Oxidative DNA Damage and Increase Resistance to Radiation(A) DLD-1 (KRASG13D) and RPE1 cells were infected with retroviruses expressing either nothing (Vector), C1C2-NLS or OGG1. Nuclear extracts were analyzed by immunoblotting using the indicated antibodies. (B) Map showing the evolutionarily conserved domains of CUX1:CC, coiled-coil; C1, C2, C3, CUT domain 1, 2 and 3; HD, homeodomain. The CUT domain 1 and 2 (C1C2) construct includes a nuclear localization signal (NLS). Diagrammatic representation of the 8-oxoG cleavage assay using a fluorophore reporter probe. Red star, 8-oxoguanine; green circle, FAM fluorophore; black circle, dabcyl quencher. (C) 8-oxoG cleavage assay was performed using whole cell extracts from DLD-1 and RPE1 cells stably carrying vectors expressing either OGG1, CUT domains 1 and 2 (C1C2) or nothing (vector). Two controls are shown. “Probe Alone” is the probe incubated in the absence of cell extract. “DLD1+normal G” is a reaction with DLD1 cell extract and a probe that contains a normal guanine residue instead of 8-oxoG; the lack of fluorescence confirms that that the increase in fluorescence intensity in the reactions with the 8-oxoG probe is not caused by a nonspecific nuclease activity. (D) 8-oxoG cleavage assay was performed using 50 nM OGG1 and 200 nM of BSA or recombinant CUT domains 1 and 2 (C1C2). A coomassie blue stain of the C1C2 purified protein is shown. (E) Cells were exposed to radiation and then submitted to a clonogenic assay. Cloning efficiency of unexposed cells was set at 100%. Results of triplicate experiments are shown. Error bars represent standard error. ***p < 0.001; **p < 0.01.; *p < 0.05; Student's t-test. (F) Cloning data in Figure 5E is represented as line graphs where non-irradiated cells were set at 100%: control cells (blue), cells overexpressing C1C2 (red) or OGG1 (green). Results of triplicate experiments are shown. Error bars represent standard error. ***p < 0.001; **p < 0.01.; *p < 0.05; Student's t-test. (G) RT-PCR analysis was performed to measure mRNA levels of genes involved in base excision repair. All mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The values are the mean of three measurements and error bars represent standard deviation.