U2OS cells were routinely maintained in DMEM (21885025, Thermo Fisher) supplemented with 10% (v/v) Fetal Bovine Serum (PAA), Penicillin (100 U/ml) and Streptomycin (100 µg/ml). A549 and SH-SY5Y were grown in DMEM (31966021, Thermo Fisher) while HMLE-E have been maintained in 1:1 Dulbecco’s Modified Eagle Medium (DMEM)/HAMF12 medium supplemented with 10% fetal calf serum, penicillin, streptomycin, amphotericin B (Life Technologies), 10 ng/mL human epidermal growth factor (EGF) (Sigma), 0.5 pg/mL hydrocortisone (Sigma) and 10 pg/mL insulin (Sigma).

For the generation of U2OS OGG1 knockout cell lines two target sequences were chosen (in exons 2 and 3), according to the web-based CRISPR design tool CRISPick (https://portals.broadinstitute.org/gppx/crispick/public). The sgRNA oligos targeting exon 2 (CAC​CGC​TCA​ACT​GTA​TCA​CCA​CTG) or exon 3 (CAC​CGA​AAG​AGA​AAA​GGC​ATT​CGA​T) were introduced into pX458 expressing Cas9 nuclease fused to GFP. pSpCas9 (BB)-2AGFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138). U2OS WT cells were transfected with the guide containing plasmid using the transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s protocol. Single GFP positive cells were sorted into 96-well plates using the BD Influx sorter (BD Biosciences). The knockout cell lines grown from the single cells were screened by western blot using an antibody against OGG1 (Abcam, ab124741).

OGG1 inhibitors TH5487 (M9506) and SU0268 (HY-139056 (MedChemExpress); 10-4700 (Focus Biomolecules) and MBS5765223 (MyBiosource)) were purchased from Clinisciences. Efflux pump inhibitors Verapamil (V4629), Fumitremorgin C (344847) and Zosuquidar/LY-335979 (SML1044) were purchased from Merck. The dyes Picogreen (P7581), Mitotracker green (M7514) and TMRE (T669) were obtained from Thermo Fisher and SiR-DNA (SC007) from SpiroChrome. The drugs Nocodazole (M1404), Etoposide (E1383) and KBrO₃ (309087) and Mitoxantrone (M6545) were purchased from Sigma.

For the quantification of γH2AX foci wild-type and OGG1-KO U2OS cells were resuspended in culture medium and seeded into an optical 96-well culture plate (89626, Ibidi) at a final density of 4000 cells/well. Plates were then incubated for 3 days at 37°C, 5% CO2. The day of the experiment, cells were treated with the indicated drugs at the indicated concentrations, and plates were returned at 37°C for 1 h. Supernatants were then removed and cells were fixed by adding 100 µL of a 4 % formaldehyde solution (w/v, 47608, Sigma) diluted in PBS with Ca2+/Mg2+ (D8662, Sigma). After 20 min, formaldehyde was removed and wells were washed three times with 200 µL PBS without Ca2+ & Mg2+ (D8537, Sigma). Cells were permeablized 10 min at RT with 150 μl of Triton-X100 (X100, Sigma) 0.5% (v/v) in PBS without Ca2+ & Mg2+. Wells were washed three times with 200 µl of PBS without Ca2+ & Mg2+, and non-specific epitopes were saturated for 60 min at RT with 150 µl of PBS + 2% BSA (w/v, A5611, Sigma) + Tween20 0.1% (v/v, P9416, Sigma). After blocking buffer removal, 100 µl of anti- γ-H2AX mouse antibody (JBW301 clone, 05-636-I, Sigma) diluted at 1:1000 in PBS + 1% BSA (w/v) + Tween 0.1% (v/v) were layered into the wells. Plates were incubated at room temperature for 90 min, then wells were washed three times with 200 µl of PBS without Ca2+ & Mg2+. 100 µl of Goat anti-Mouse antibody coupled to Alexa-488 diluted at 1:1000 in PBS + BSA 1% (w/v) + Tween 0.1% (v/v) were layered into the wells. Plates were incubated at room temperature for 90 min, then wells were washed three times with 200 µl of PBS without Ca2+ & Mg2+. Nuclear DNA was then probed 20 min with 150 µl of Hoechst 33342 (#B2261, Sigma) diluted at 2 μg/mL in PBS without Ca2+ & Mg2+. Wells were then washed two times with 200 µl of PBS without Ca2+ & Mg2+, and 200 µl of PBS were added to the wells. Plates were sealed with an aluminium foil and imaged on a high-content imaging microscope (Operetta, Perkin Elmer) at ×20 and ×40 magnification (13 fields acquired/well) in the blue (Ex. 360–400 nm, Em. 410–480 nm) and green (Ex. 460–490 nm, Em. 500–550 nm) channels. Using a high-content imaging analysis software (Harmony 3.0, Perkin-Elmer), nuclear ROI were segmented in the Hoechst (blue) channel and the number of γ-H2AX foci per nuclei was quantitated in ×40 magnification images using a foci-detection algorithm.

For visualization of 8-oxoG, HeLa OGG1–GFP cells grown on 8-well glass bottom chamber slides (80827, ibidi) were fixed in acetone:methanol (1:1) and air dried. Cells were hydrated for 15 min in PBS, and DNA was denatured by incubating cells in 2N HCl for 45 min at room temperature. Cells were washed three times in PBS, neutralized with 50 mM Tris–HCl pH 8.8 for 5 min and permeabilized at room temperature in PBS–0.1% Triton for 10 min. Cells were incubated in blocking solution (PBS, 0.1% Triton, 3% BSA, 1% normal goat serum) at 37°C for 1 h and subsequently incubated for 1 h at 37°C with antibodies against 8-oxoG (ab48508, abcam) and GFP (632592, Clontech) diluted in blocking solution at 1:2000 and 1:1000 respectively. Cells were then washed three times for 10 min in PBS–.1% Triton and incubated with an anti-Mouse antibody coupled to Alexa-488 (A11001, Thermofisher) and an anti-Rabbit antibody coupled to Alexa-647 (A31573, Thermofisher), both diluted at 1:1000 in blocking solution for 1 h at 37°C. Nuclear DNA was counterstained with 1 μg/ml propidium iodide with 50 μg/ml RNAse. Cells were imaged with a Nikon A1 confocal microscope, using a ×60 oil immersion objective with a numerical aperture of 1.3 and analysis was performed with Fiji (Schindelin et al., 2012).

For stainigs with Picogreen and Nuclight, wild-type and OGG1-KO U2OS cells were resuspended in culture medium and seeded into the wells of an optical 96-well culture plate (#3904, Costar) at a final density of 2000 cells/well. Plates were then incubated for 3 days at 37°C, 5% CO2. The day of the experiment, cells were first incubated 30 min at 37°C with Picogreen (1:500 [V/V] dilution from the supplier’s stock solution, Invitrogen). Supernatants were then removed and wells were washed once with 150 µL PBS with Ca2+/Mg2+. 50 µl of a suspension of complete medium containing Nuclight (1:500, v/v, #4717, Sartorius) alone or mixed together with the indicated inhibitors were layered into the culture wells. After 1 h of incubation at 37°C, supernatants were removed and replaced with 150 µl of complete medium. Plates were immediately imaged on a high-content imaging microscope (Operetta, Perkin Elmer) at ×10 magnification (9 fields acquired/well) in the green (Ex. 460–490 nm, Em. 500–550 nm) and far red (Ex. 620–640 nm, Em. 650–760 nm) channels. Using a high-content imaging analysis software (Harmony 3.0, Perkin-Elmer), Picogreen fluorescence was used to segment a nuclear Region of Interest (ROI), in which the average Nuclight fluorescence intensity was quantitated.

For DNA staining with Hoecsht 33342, cells grown on 8-well glass bottom chamber slides were incubated for 5 min at 37°C in medium containing the dye at 1 μg/ml, washed twice in warm medium and immediately imaged with a spinning disk microscope (GATACA, W1) using a ×60 oil objective and an excitation laser at 405 nm.

Cells were seeded at 10.000 cells/well in 96 well plates and 3 days after seeding cells were incubated for 1 h in DMEM containing either DMSO (NT), or the OGG1i TH5487 or SU0268 at a concentration of 10 µM. Cells were further stained for 20 min with 100 nM of TMRE and 100 nM of MitoTracker Green in the presence of OGG1i and fluorescence was immediately quantified using a microplate reader (CLARIOstar—BMG Labtech).

Cells were trypsinized and resuspended to a final concentration of 2 million cells/mL. For each condition, 400′000 cells (200 µl) were distributed in flow cytometry tubes and OGG1i or pump inhibiting drugs were added at a 2X of final concentration. On top of that, 200 µl of warm medium containing the investigated dye at 2X of final concentration (MitoTracker Green 100nM, TMRE 100nM, Sir-DNA 333nM, Hoechst33342 0.1 μg/μl, and Mitoxantrone 1 µM) was added. Cells were incubated 30 min at 37°C and further 10 min at room temperature for equilibration before flow cytometry analysis with the NovoCyte Flow Cytometer System (Agilent).

The immunostaining for Phospho-H3 was performed in cells resuspended, fixed and permeabilized in ice-cold 75% ethanol. Upon washing, cells were blocked in PBS buffer containing 1% BSA, 0.05% Triton X100 and cells were incubated overnight at 4°C with an Pacific Blue Conjugate monoclonal antibody against Phospho-Histone H3 (Ser10) (8552, Cell Signaling) diluted at 1:500 in PBS buffer containing 1% BSA, 0.05% Triton X100. Washed cells were resuspended in PBS-1% BSA in the presence of 0.3 µM of Topro-3 for DNA staining 1 h at room temperature before FACS analysis with the NovoCyte Flow Cytometer System (Agilent).

Cell pellets (from 3 × 10exp6 cells) were resuspended in 50 µl of Lysis buffer (Tris 20 mM, NaCl 20 mM, SDS 0.1%, MgCl₂ 1 Mm, Benzonase 0.25 U/µl and Protease inhibitors cocktail 1X) and sonicated for 10 min (with pulses 30 s on/30 s off). After sonication the samples were centrifuged at 12000 at 4°C for 5 min and the supernatants were recovered. Protein concentration was measured with the Bradford assay (Bio-Rad #500-0006), laemmli buffer was added at a 1x concentration (0.1% 2-mercaptoethanol, 0.0005% Bromophenol blue, 10% glycerol, 2% SDS, and 63 mM Tris pH 6.8) before heating for 5 min at 95°C, and 20 µg of protein extracts were loaded in a mini PROTEAN TGX stain-free gel (Bio-Rad #4568083). Precision plus protein dual color standards (Bio-Rad #1610374) was used as a Molecular weight marker. The transfer on nitrocellulose membrane (Bio-Rad #1704157) was performed with the Trans-Blot^® Turbo™ Transfer System (Bio-Rad #1704150). The membrane was blocked for 1 h in blocking solution (PBS-0.1% Tween20 containing 5% milk), and incubated for 1 h at room temperature with primary antibodies (anti-OGG1 Ab124741 1:10000 and Anti-Vinculine Ab18058 1:4000). Membranes were washed three times for 5 min with PBS-0.1% Tween20 and further incubated for 45 min in secondary antibodies anti-rabbit IR800 (Diagomics R-05060) and anti-mouse IR700 (Diagomics R-05055) diluted at 1/10.000 in PBS-0.1% Tween 20 containing 5% milk. Western blots were imaged with the Li-Cor Odyssey DLx system.

RNA was extracted from 1 × 10 exp6 cells using RNeasy Plus micro kit (#74034, QIAGEN) following manufacturer instructions and the concentration measured with Nanodrop 2000c (Thermo Fisher Scientific). 300 ng of RNA was used for the reverse transcription with the Thermo-Fisher Super Script Vilo kit (#11754050) using the following cycle: 1 min at 25°C, 60 sec at 42°C and 5 sec at 85°C in Eppendorf thermocycler (Eppendrof Mastercycler ep gradient S). Resulting cDNAs were amplified by qPCR with the iTaq Universal Probes Supermix (Biorad #1725130, Biorad) using the Applied Biosystems™ QuantStudio™ three Real-Time PCR System. Taqman probes against MDR1 (Hs00184500_m1), BCRP1 (Hs01053790_m1), and GAPDH (HS9999905-m1) were purchased from Thermo Fisher Scientific.

Vesicular transport inhibition assays were carried out by SOLVO^® Biotechnology. Briefly, vesicles from HEK293 cells overexpressing MDR1, BCRP or MRP1 were purified to have an inside-out pump configuration. The reaction mix containing the vesicles, the specific corresponding radiolabeled substrate [N-methyl-quinidine (NMQ), Estrone-3-sulfate (E3S) or β-estradiol-17-β-D-glucuronide (E217β)], and specific corresponding pump inhibitor [Valspodar (1 μM), Ko143 (0.2 μM) or Benzbromarone (200 μM)), OGG1i (TH5487 and SU0286 (10 and 100 μM)] or DMSO were resuspended in transport buffer and incubated for 15 min at 37°C for MRP1 and 32°C for MDR1 and BCRP. The start reagent solution, containing a final concentration of either 4 mM Mg-ATP or 4 mM AMP in transport buffer, was added on top of the reaction mix. The reactions were incubated for 1 min for BCRP and MDR1 and for 3 min for MRP1 and quenched by the addition of ice-cold washing mix. The samples were transferred to a filter plate and washed 5-times with ice-cold washing mix, and the amount of probe substrate inside the filtered vesicles was quantified by liquid scintillation counting and expressed as counts per million (cpm). For each studied condition, the cpm values in the presence of ATP were subtracted by the corresponding values in the presence of AMP (passive diffusion of the substrate inside the vescicles, background signal). The resulting values have been normalized to DMSO and expressed in % as a measure of pump inhibition effect for each tested drug.

For the proliferation curves, 1000 cells resuspended in 75 µl culture complete media were seeded in a 96 well plate. After 6 h, 25 µl of culture medium containing or not OGG1i at 4X of final concentration were added. Cell proliferation was followed for 1 week with the Incucyte^® S3 System by acquiring five pictures per well at an image rate of one image every 3 h. The resulting transmission images were analysed with the Incucyte^® S3 segmentation algorithm generating a coverage mask used to follow cellular confluence.

To evaluate cellular sensitivity to Etoposide treatments, wild-type and OGG1-KO U2OS cells were resuspended in culture medium and seeded into the wells of an optical 96-well culture plate (#3904, Costar) at a final density of 1000 cells/well. Plates were incubated 24 h at 37°C, 5% CO2 to allow for cell adhesion. 24 h post-seeding, cells were treated with the indicated drugs at the indicated concentrations for 1 h at 37°C, washed with fresh medium and incubated for 96 h at 37°C, 5% CO2. At the end of the incubation period, cells were fixed with formaldehyde 4% solution, diluted in PBS with Ca2+/Mg2+ containing Hoechst 33342 at 2 μg/mL. After an overnight incubation at 4°C in the dark, fixation solution was removed by aspiration, replaced by 150 µl of PBS with Ca2+ and Mg2+, and plates were imaged on a high-content imaging microscope (Operetta, Perkin Elmer) at ×10 magnification (9 fields acquired/well) in the blue channel (Ex. 360–400 nm, Em. 410–480 nm). Using a high-content imaging analysis software (Harmony 3.0, Perkin-Elmer), DNA-labeled nuclei were segmented, and the absolute amount of nuclei per condition was quantitated. Results were expressed as the amount of cells relative to the average amount of cells in the untreated wells.

Statistical analysis were performed with FlowJo or GraphPad Prism 9.1.2 softwares. The statistical test used is indicated in the Legend to the Figures.

In our aim to evaluate the impact of OGG1i in both nuclear and mitochondrial function, U2OS cells exposed or not to OGG1i were stained with the mitochondrial probes Mitotracker green and TMRE, indicators of mitochondrial mass and activity respectively. Surprisingly, the levels of fluorescence obtained were significantly higher when the staining was done in the presence of OGG1 inhibitors TH5487 and SU0268 (Figure 1A). Similar observations were made with the cell permeant DNA probes SiR-DNA and NucLight, classically used for nuclear staining in living cells. While NucLight very slightly stained the nucleus of U2OS, staining was again much stronger in the presence of the two OGG1 inhibitors (Figure 1B). It is known that those dyes can be exported by the efflux pumps and that’s the reason why they are often combined with the general efflux pump inhibitor Verapamil in order to improve cellular staining (Lukinavičius et al., 2015). As expected, the incubation of cells with NucLight in the presence of Verapamil resulted in a much stronger nuclear staining that was very similar to the one obtained with the OGG1 inhibitors SU0268 and TH5487. To better quantify the observed effects, cells were simultaneously stained with another DNA-intercalator, Picogreen, prior to the incubation with NucLight. Picogreen staining was used for the segmentation of the nucleus and the generation of a mask that was applied to the NucLight image in order to quantify the nuclear staining. A significant increase in the NucLight staining was observed in the presence of TH5487 and SU0268, again similar to the observed with the efflux pump inhibitor Verapamil (Figure 1D and Supplementary Figure S1). Exactly the same effects were observed in two independent U2OS cell lines in which OGG1 was knocked out by CRISPR-Cas9 using guideRNAs targeting exon 2 (KO2) or exon 3 (KO3) (Figures 1C, D). These results indicate that the observed effects of the DNA glycosylase inhibitors were not dependent on OGG1 but necessarily an off-target effect.

Dose response curves using different concentration of OGG1 inhibitors were performed by flow cytometry analysis using SiR-DNA (Figure 1E) and TMRE (Figure 1F) and clearly showed that the observed effects were dependent on the concentration of OGG1 inhibitors. Here again, the same observations were made in WT cells and in cells deficient for OGG1 confirming the off-target effect (Figures 1E, F).

Considering the unexpected effects of OGG1 inhibitors reported here, we decided to evaluate if those molecules were still acting as OGG1 inhibitors in our experimental conditions. With that purpose, we determined the repair kinetics of 8-oxoG following exposure of cells to KBrO3, a drug known to induce large amounts of 8-oxoG in genomic DNA. Cells overexpressing OGG1-GFP were used in this experiment in view of their faster repair kinetics compared to the cells expressing OGG1 at endogenous levels (Amouroux et al., 2010; Lebraud et al., 2020; Supplementary Figures S2A–C). As shown in Supplementary Figures S2C,D, repair of the 8-oxoG was clearly impaired in cells incubated in the presence of OGG1 inhibitors TH5487 and SU0268. Therefore, we concluded that despite the capacity of these molecules to inhibit the enzymatic activity of OGG1, they show off-target effects at the cellular level that are independent on OGG1.

Since the effects observed for the OGG1 inhibitors TH5487 and SU0268 were very similar to the ones observed with the general efflux pump inhibitor Verapamil, we wondered if OGG1 inhibitors could have a direct effect on efflux pumps. Many different efflux pumps have been identified in human cells, and cancer cell lines express a combination of several of them, being BCRP, MDR1, and MRP1 the most widely overexpressed ones (Wang et al., 2021; Robey et al., 2018). In order to evaluate the direct effect of OGG1 inhibitors on efflux pump activity, we used the vesicular trafficking in vitro assay. The principle of the assay relies on measuring the accumulation of a substrate into vesicles containing the efflux transporter in an inside out orientation, with the ATP and substrate binding sites of the transporter exposed to the buffer outside. By utilizing inside out vesicles, the ATP-dependent transport of substrate is directed into the vesicular lumen, where low permeability compounds are then essentially trapped. HEK293 cells are particularly appropriated for this kind of analysis as they express moderate levels of efflux pumps and allow to evaluate the effect of drugs on a individually overexpressed efflux pumps. Thus, vesicles were prepared from HEK293 cells overexpressing either BCRP, MRP1 or MDR1. Vesicles were incubated with the radiolabeled molecules E3S, NMQ or E₂17β, that are specific substrates for BCRP, MDR1, and MRP1 respectively, in the presence of ATP (active transport) or AMP (background). The amount of radioactive substrate taken up into the vesicles in an ATP dependent manner, was quantified by liquid scintillation counting inside the filtered vesicles (illustration Figure 2A). Specific inhibitors for each of the evaluated pumps (Ko143 for BCRP, valspodar for MDR1, and Benzbromarone for MRP1) were used as positive controls in the assay. At the dose of 10 μM, classically used in cellulo experiments using OGG1 inhibitors, TH5487 showed an almost complete inhibitory effect on BCRP (99% inhibition), and a mild effect on MDR1 (27%) or MRP1 (8%). SU0268 inhibited both BCRP and MDR1 at 76% and showed a milder but still significant effect on MRP1 (15% inhibition) (Figure 2B). At a concentration of 100 μM, both OGG1 inhibitors show an inhibitory effect on the three efflux pumps analysed.

The identification of BCRP and MDR1 as direct targets of OGG1 inhibitors could explain the results presented in Figure 1 as both SiR-DNA, Mitotracker green and TMRE are known substrates of those efflux pumps (Lukinavičius et al., 2015; de Almeida et al., 2017; Mansell et al., 2021). In order to evaluate if the effects observed for OGG1 inhibitors were indeed dependent on the levels of expression of the efflux pumps we selected different cancer cell lines according to their respective expression levels of BCRP and MDR1. Based on the information available in the Human Protein Atlas (https://www.proteinatlas.org/) we decided to evaluate the effect of OGG1 inhibitors on A549 cells expressing high levels of BCRP, SH-SY5Y cells expressing high levels of MDR1, and HMLE showing very low expression of both efflux pumps. The levels of expression of BCRP and MDR1 genes were quantified in the different cellular models by RT-qPCR, and were in agreement with the data available in the Human Protein Atlas (Figure 2C). Consistently with the results obtained with the vesicular traffic assay, the effect of both TH5487 and SU0268 on the fluorescent signal obtained upon staining with SiR-DNA, a good substrate of MDR1 (Lukinavičius et al., 2015; Sen et al., 2018), was significantly stronger in SH-SY5Y cells, compared to the effects observed in HMLE cells in which MDR1 expression is undetectable (Figure 2D). We next monitored the effect of OGG1 inhibitors on fluorescent levels obtained after staining the cells with HO33342, a good substrate of BCRP (Abbott, 2003). We observed a very strong effect of the OGG1 inhibitor TH5487 on the fluorescent signal of HO33342 in A549 cells, expressing higher levels of BCRP. Interestingly, the effect of TH5487 was very similar to the observed with Fumitremorgin C, a specific and strong inhibitor of BCRP. A weaker but clearly detectable effect, was observed with the OGG1 inhibitor SU0268, that was comparable with the effect observed with the very well-known and highly used efflux pump inhibitor Verapamil (Figure 2E). These results are in agreement with those obtained in the vesicle transport assay showing that BCRP activity was inhibited at 100% by TH5487 and at 76% by SU0268. Almost no effect of TH5487 or SU0268 could be observed in HMLE cells, expressing BRCP1 at undetectable levels (Figure 2E). Altogether, these results clearly indicate a positive correlation between the level of expression of efflux pumps and the effect of OGG1 inhibitors on the accumulation of fluorescent probes inside the cells.

Not only fluorescent probes are transported by the efflux pumps but also several chemotherapeutical drugs. Indeed, many cancer cells overexpress efflux pumps allowing them to export drugs out of the cells, a phenomenon at the origin of the observed drug multiresistance in several cancers (Wang et al., 2021; Robey et al., 2018). To evaluate if OGG1 inhibitors could influence the intracellular concentration of chemotherapeutical drugs, we incubated the cells with Mitoxantrone (MTX), a Topoisomerase II inhibitor that emits a fluorescence signal and is a known substrate of BCRP (Abbott 2003). The red fluorescence emitted by MTX was measured by flow cytometry and, as observed for the other fluorescent dyes used so far in this study, higher fluorescent levels were detected in cells simultaneously incubated with MTX and the OGG1 inhibitors TH5487 and SU0268 (Figure 2F). Again, the effects of OGG1 inhibitors where comparable to the ones observed with efflux pumps inhibitors Verapamil and Fumitremorgin (Figure 2F). If the increase in the intracellular levels of MTX observed in the presence of OGG1 inhibitors is due to their inhibitory effect on efflux pumps, we could expect OGG1 inhibitors to potentiate the effect of many chemotherapeutical drugs known to be substrates of BCRP1 or MDR1 such as doxorubicin, paclitaxel, cisplatine, 5-FU, etoposide, (Wang et al., 2021). Indeed, a higher number of γH2AX foci, used as an indicator of double strand breaks (DSB), were induced by Etoposide treatment in the presence of OGG1 inhibitors TH5487 and SU0268. The fact that the same effects were observed in OGG1 deficient cells, clearly points out an off-target effect and discards the involvement of OGG1 activity (Figures 3A–C). In agreement with the higher level of DNA damage observed, cell survival was also affected, with higher mortality observed in cells treated simultaneously with Etoposide and OGG1 competitive inhibitors, compared to the ones exposed to the single treatment with Etoposide (Figure 3D). The synergistic effect observed between Etoposide and OGG1 inhibitors is independent of OGG1 activity and could be explained by the off-target effect of both TH5487 and SU0268 on efflux pumps.

In order to evaluate if OGG1 inhibitors had an effect on cell proliferation and if it was dependent on OGG1, TH5487 or SU0268 were added to the culture medium at a concentration of 10 µM and cellular proliferation was monitored by using the Incucyte^® Live-Cell Analysis System. No significant effects could be observed in the presence of TH5487, and although cell proliferation was slightly slowed down, the effects were not dependent on OGG1 since very similar behaviour was observed between WT and OGG1 KO cells. In contrast, when 10 µM of SU0268 was added to the cell culture medium a dramatic proliferation arrest was observed for both WT and OGG1 KO cells (Figure 4A). The same effects were observed with three independent batches of SU0268 purchased from independent suppliers (see material and methods for references). The images obtained in this assay unveiled a large number of cells showing a round shape characteristic of cells undergoing mitosis (Figure 4B). Indeed, staining of genomic DNA with HO33342 revealed that both U2OS WT and OGG1 KO cells incubated in the presence of SU0268 had a defect on mitotic progression as all mitotic cells were blocked at the prophase step of the cell cycle, and no cells progressing through Metaphase, Anaphase or Telophase could be observed (Figure 4C). The mitotic figures observed in the presence of SU0268 were comparable to the ones induced by incubating the cells with Nocodazole, a molecule known to disrupt microtubule assembly/disassembly dynamics, and thus impairing formation of the metaphase spindles during the cell division cycle (Lara-Gonzalez et al., 2012). The percentage of cells undergoing mitosis was quantified by flow cytometry using an antibody specific against the mitotic marker phospho-Histone H3 (Ser10). While the level of mitotic cells was around 2% in cells grown in normal culture medium or in the presence of TH5487, up to 16% of mitotic cells accumulated 6 hours after the addition of 10 µM of SU02568 in the medium (Figure 4D). It is important to note that effects on cell cycle progression were not cell line dependent as very similar results were obtained in different cell lines such as HMLE, A549, and T47D (Supplementary Figure S3).