*4.7. Indirect Inmunocytofluorescence and Image Acquisition*

Cells were washed with filtered PBS (fPBS, 0.22 µm pore size), fixed in 4% paraformaldehyde (PFA) in fPBS 15 min at 4 ◦C, washed in fPBS, permeabilized in 0.1% Triton-X100 in fPBS, and immersed in blocking buffer (0.2% Tween, 1% BSA in fPBS) for 30 min. Briefly, cells were incubated with the specific antibodies, namely, 1:300 rabbit anti-PARP-1/2 (Santa Cruz sc-7150, CA, USA), 1:500 rabbit anti-PAR (BD551813, Becton Dickinson (Franklin Lakes, NJ, USA)), 1:50 mouse anti-PAR (Enzo BML-SA216, Farmingdale, NY, USA), 1:100 10H-anti-PAR (Tulip #1020), 1:200 anti-hPARG (Abcam 16060, Cambridge, MA, USA), 1:400 mouse monoclonal anti-γH2AX (Abcam), and 1:300 anti-53BP1 (Abcam 36828), and diluted in blocking buffer for 2 h at 37 ◦C. After washing in fPBS/T (0.1% Tween), cells were incubated (1 h, RT) with the correspondent anti-antibodies mix (1:250 anti-mouse-Cy3 Jackson Immuno Research, 1:1000 goat-anti-rabbit 488 (#A-11034, Thermo Fisher Scientific, Waltham, MA, USA) in a blocking buffer. After washing in fPBS/T and fPBS, DAPI counterstaining (1.5 µg/mL in fPBS), and a final wash in fPBS, the coverslips were mounted in Prolong Gold (Molecular Probes P36930, Eugene, OR, USA) and sealed with nail polish. Controls without a primary antibody were run in parallel to check the specificity of the signals.

Mainly an Olympus BX61/FV300 (Tokyo, Japan) and sometimes a Zeiss LSM800-Airyscan (Oberkochen, Germany) or a Leica microscope were used to take the confocal images/stacks. Fluorescence excitation was performed with the following lasers: diode 405 nm (DAPI), multiline argon 488 nm (Alexa Fluor 488), and helium-neon 561 nm (Cy3). Microscope settings were adjusted to register no signal in controls without primary antibodies. The scanning of optical sections was performed sequentially for the different fluorochromes. All images in each experimental series were taken with the same setting at the same confocal session. If modified, all were subject to the same degree of brightness/contrast adjustment and Gaussian blur filtering, including the control without a primary antibody. ImageJ free software [83] was used for the image processing.

#### *4.8. Cell Counting Using Low-Magnification Fields*

The ImageJ "Cell Counter" plug-in [84] (was employed to find out the percentage of nuclei that exhibited no γH2AX, well-defined γH2AX foci, or γH2AX pan-nuclear staining.

#### *4.9. Relative PAR Quantification*

The total field PAR intensity (RawIntDen, the sum of signal intensity in all the image pixels) was normalized using DAPI RawIntDen in each field (to account for putative differences in cell densities). Then, the data were expressed as a percentage of the control.

### *4.10. Statistical Analysis*

The results were expressed as mean ± SEM. Differences between different experimental groups were tested for significance using a two-tailed unequal variances Student's *t*-test in Microsoft Excel 2007 [85] or a one-way analysis of variance (ANOVA), followed by post-hoc multiple comparisons tests (Tukey, Scheffe, Bonferroni, and Holm) in ASTATSA [86])

**Author Contributions:** Conceptualization, V.P., M.S., P.L., D.J.K., and L.L.-H.; methodology, V.P., M.S., P.L., S.V.-L., and L.L.-H.; formal analysis, V.P., M.S., P.L., S.V.-L., D.J.K., and L.L.-H.; investigation, V.P., M.S., P.L., S.V.-L., D.J.K., and L.L.-H.; resources, V.P., M.S., P.L., S.V.-L., D.J.K., and L.L.-H.; data curation, V.P., M.S., P.L., S.V.-L., D.J.K., and L.L.-H.; writing—original draft preparation, L.L.-H.; writing—review and editing, V.P., M.S., P.L., S.V.-L., D.J.K., and L.L.-H.; visualization, L.L.-H.; supervision, D.J.K; project administration, D.J.K.; funding acquisition, V.P., M.S., D.J.K., and L.L.-H. All authors have read and agreed to the published version of the manuscript (CRediT taxonomy).

**Funding:** This research was partially funded by an undergraduate Student Research Support Program, call 2017: "Programa de Apoyo a la Investigación Estudiantil, Comisión Sectorial de Investigación Científica" (PAIE-CSIC UdelaR, Uruguay). Project title: Poly(ADP-ribosylation) and VERO cells' response to induced DNA damage. Responsible student: V.P.; responsible professor: D.J.K; co-responsible: L.L.H. Some reagents were bought through

Programa de Desarrollo de las Ciencias Básicas (PEDECIBA). APC contributors: PEDECIBA BIOLOGIA (aliquots given to researchers D.J.K., L.L.-H and R. Daniel Peluffo, CENUR); and Ministerio de Educación y Cultura.

**Acknowledgments:** Agencia Nacional de Investigación e Innovación (ANII-SNI). Bleomycin sulfate (Bleocris 15, LKM) was a gift from Laboratorios Teva Uruguay S.A., Grupo Biotoscana. The single-cell electrophoresis chamber and power supply were from the Epigenetics and Genomic Instability Lab, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE). The alternative CO<sup>2</sup> incubators used came from the Neurochemistry Department, IIBCE, and the Microbiology Department, IIBCE. Part of the VERO cell stock was kept at Facultad de Ciencias, UdelaR. G. A. Folle kindly contributed to the text editing. Thanks to R.D. Peluffo and to Ministerio de Educación y Cultura, Dirección para el Desarrollo de la Ciencia y el Conocimimento, for financial contributions to pay APC.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
