**Abbreviations**



RT Room temperature SSBs Single-strand breaks

UV-C Ultraviolet C radiation

VRK1 Vaccinia-related kinase 1

Western Blots

TNKS Tankyrase

#### **Appendix A Appendix A**

WB

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 16 of 23

VE-821 3-amino-6-(4-methylsulfonylphenyl)-N-phenylpyrazine-2-carboxamide (ATRi)

VE-822 3-[3-[4-(methylaminomethyl)phenyl]-1,2-oxazol-5-yl]-5-(4-propan-2-

ylsulfonylphenyl)pyrazin-2-amine (ATRi)

VERO African green monkey kidney epithelial cell line

**Figure A1.** BLEO and OLA curves. Cell viability (MTT assay). (**A**) Cells were exposed to BLEO in the indicated doses (0–160 µg/mL) during a 45 min pulse. Then, cells were cultured during the indicated time before the MTT addition (0, 24, or 72 h, where "t = 0" refers to immediately after the treatment). The 2018–2020 dataset includes 20 MTT experiments with BLEO in VERO cells. At 72 h, *n* = 63, 6, 72, and 22 for increasing BLEO concentrations. Fewer data were recorded for shorter times and are depicted to facilitate understanding of the chosen schedule.\*\*\*: p < 0.001. (**B**) Cell viability (24 h) under higher BLEO doses or longer BLEO pulse durations. Cells were exposed to BLEO in the indicated doses (0–500 µg/mL) during a 45 min pulse or (last bar) during 270 min. (**C**) Cell viability (24 h) in the control and BLEO-exposed cells in the absence or presence of a co-treatment (45 min pulse) with bleomycin hydrolase inhibitor E64. (**D**) Cell viability after exposure to OLA (0–400 nM). The whole curve was done once at 24 h and once at 72 h. In contrast, for 50 nM Olaparib at 72 h, *n* = 75 from 12 independent experiments. **Figure A1.** BLEO and OLA curves. Cell viability (MTT assay). (**A**) Cells were exposed to BLEO in the indicated doses (0–160 µg/mL) during a 45 min pulse. Then, cells were cultured during the indicated time before the MTT addition (0, 24, or 72 h, where "t = 0" refers to immediately after the treatment). The 2018–2020 dataset includes 20 MTT experiments with BLEO in VERO cells. At 72 h, *n* = 63, 6, 72, and 22 for increasing BLEO concentrations. Fewer data were recorded for shorter times and are depicted to facilitate understanding of the chosen schedule. \*\*\*: *p* < 0.001. (**B**) Cell viability (24 h) under higher BLEO doses or longer BLEO pulse durations. Cells were exposed to BLEO in the indicated doses (0–500 µg/mL) during a 45 min pulse or (last bar) during 270 min. (**C**) Cell viability (24 h) in the control and BLEO-exposed cells in the absence or presence of a co-treatment (45 min pulse) with bleomycin hydrolase inhibitor E64. (**D**) Cell viability after exposure to OLA (0–400 nM). The whole curve was done once at 24 h and once at 72 h. In contrast, for 50 nM Olaparib at 72 h, *n* = 75 from 12 independent experiments.

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 17 of 23

#### **Appendix B Appendix B**

**Appendix C** 

**Figure A2.** Concentrations of 150 nM OLA and BLEO. A single experiment was carried out to explore whether an increase in OLA concentration would lead to potentiation. Effect of combined 150 nM OLA and 40 µg/mL BLEO in the absence (**A**) or presence (**B**) of DMSO on VERO cell viability was evaluated using MTT (72 h). Mean ± SD. *n* = 6. Since the predicted additive effect according to Webb's equation [25] was equal or lower than the combined effect, synergism was discarded. **Figure A2.** Concentrations of 150 nM OLA and BLEO. A single experiment was carried out to explore whether an increase in OLA concentration would lead to potentiation. Effect of combined 150 nM OLA and 40 µg/mL BLEO in the absence (**A**) or presence (**B**) of DMSO on VERO cell viability was evaluated using MTT (72 h). Mean ± SD. *n* = 6. Since the predicted additive effect according to Webb's equation [25] was equal or lower than the combined effect, synergism was discarded.

**Figure A3.** PARPis and BLEO in VERO. PARPis and BLEO in VERO. (**A**) Scheme representing PAR, its synthesis by PARPs, its degradation mainly by poly-ADP-glycohydrolase (PARG) activity, and the inhibitors abbreviations associated with the correspondent enzyme. 3AB, EB, and OLA are PARPis, while DEA inhibits PARG. (**B**) Test of the inhibitors' effects on PAR. The inhibitors used were 3 aminobenzamide (3AB, Sigma A-0788), 5′-deoxy-5′-[4-[2-[(2,3-dihydro-1oxo-1H-isoindol-4 yl)amino]-2-oxoethyl]-1-piperazinyl]-5′-oxoadenosine dihydrochloride (EB, Alexis Biochemicals), 6,9-diamino-2-ethoxyacridine-DL-lactate monohydrate (DEA, Trevigen 4680-096-03), and OLA. PAR was measured on confocal planes of monolayers using the Analyze/Measure ImageJ plugin, and the results are expressed as arbitrary fluorescence units/area. (**C**–**F**) Cell viability assays. Results are expressed as % MTT absorbance with respect to control. The violet arrows at the top of each graph represent the time in days (5 days) from seeding to MTT evaluation. The bleomycin treatment (40 to 80 µg/mL, 45 min) is represented in green and the inhibitors' treatment durations are in red. (**C**) Pretreatment with 3AB or EB, (**D**) co-treatment with 3AB or EB, (**E**) pre- and co-treatment with the PARG **Appendix B** 

**Appendix C** 
