**Appendix D**

**Appendix D** 

different from the control (*p* < 0.001). Asterisks refer to a comparison of a treatment vs. the control or

with respect to the genotoxic treatment. \*: *p* < 0.05, \*\*: *p* < 0.01.

**Figure A4.** Positive controls for anti-PAR antibodies. (**A**–**D**) Comparison of the control (**A**,**C**) and BLEO-treated cells (**B**,**D**; 40 µg/mL, 45 min, like VERO) that were indirectly immunostained with 10H anti-PAR (**A**,**B**, red) and anti-53BP1 (**C**,**D**, green) Bar: 15 µm. PAR-positive nuclei were much more frequent in CHO9 than in VERO cells. (**E**,**F**) ICF with BD anti-PAR antibody (same experiment as in Figure 3G, H, extracted from Ph.D. thesis [53] Bar: 25 µm). Only after a very strong treatment with MMS (**F**; 6 mM, 45 min), which induced cell death of the whole cell population (insert), was a slight PAR increase noticed with BD anti-PAR. (**G**) WB detecting PAR with BD anti-PAR in control cells or cells exposed to 100 and 250 µM H2O2. (**H**) WB detecting PAR indirectly with the rabbit PAR-binding reagent MABE1031 (1:4000) and anti-rabbit-HRP in control VERO cells or under exposure to 1 mM H2O2 (10 min), 8 mM MMS (45 min), or the PARP inhibitor 3AB (2 h). (**I**) WB detecting PAR with MABE1031 in the control VERO cells or under exposure to OLA (1 h), H2O2 (last 10 min), or the combined treatment OLA + H2O2. **Appendix E Figure A4.** Positive controls for anti-PAR antibodies. (**A**–**D**) Comparison of the control (**A**,**C**) and BLEO-treated cells (**B**,**D**; 40 µg/mL, 45 min, like VERO) that were indirectly immunostained with 10H anti-PAR (**A**,**B**, red) and anti-53BP1 (**C**,**D**, green) Bar: 15 µm. PAR-positive nuclei were much more frequent in CHO9 than in VERO cells. (**E**,**F**) ICF with BD anti-PAR antibody (same experiment as in Figure 3G,H, extracted from Ph.D. thesis [53] Bar: 25 µm). Only after a very strong treatment with MMS (**F**; 6 mM, 45 min), which induced cell death of the whole cell population (insert), was a slight PAR increase noticed with BD anti-PAR. (**G**) WB detecting PAR with BD anti-PAR in control cells or cells exposed to 100 and 250 µM H2O<sup>2</sup> . (**H**) WB detecting PAR indirectly with the rabbit PAR-binding reagent MABE1031 (1:4000) and anti-rabbit-HRP in control VERO cells or under exposure to 1 mM H2O<sup>2</sup> (10 min), 8 mM MMS (45 min), or the PARP inhibitor 3AB (2 h). (**I**) WB detecting PAR with MABE1031 in the control VERO cells or under exposure to OLA (1 h), H2O<sup>2</sup> (last 10 min), or the combined treatment OLA + H2O<sup>2</sup> .

**Figure A5.** γH2AX vs. 53 BP1 foci distribution in one experiment. Foci counting required the following steps, which were done in batch: (1) Separate channels. (2) Segment images to obtain a mask using a fixed threshold for all images of the experiment in each channel. In this case, the following thresholds were used: 50 for the red channel (γH2AX), 62 for the green channel (53BP1), and 40 for the blue (DAPI) channel. (3) The Analyze Particles plug-in allowed for counting objects with certain properties, namely, circularity and size. Foci were defined as ovoid to circular objects (circularity > 0.5) and 5 to 200 pixels. Nuclei were defined as ovoid objects measuring 500 to 500,000 pixels. In each case, the program showed the masks, displayed results, and summarized the data. In this way, we obtained the number of γH2AX foci, 52 BP1 foci, and cell number. (4) In order to determine the

#### **Appendix E Appendix E**

**Appendix D** 

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 18 of 23

inhibitor DEA, and (**F**) co- and post-treatment with DEA. The genotoxic treatment was always different from the control (*p* < 0.001). Asterisks refer to a comparison of a treatment vs. the control or

**Figure A4.** Positive controls for anti-PAR antibodies. (**A**–**D**) Comparison of the control (**A**,**C**) and BLEO-treated cells (**B**,**D**; 40 µg/mL, 45 min, like VERO) that were indirectly immunostained with 10H anti-PAR (**A**,**B**, red) and anti-53BP1 (**C**,**D**, green) Bar: 15 µm. PAR-positive nuclei were much more frequent in CHO9 than in VERO cells. (**E**,**F**) ICF with BD anti-PAR antibody (same experiment as in Figure 3G, H, extracted from Ph.D. thesis [53] Bar: 25 µm). Only after a very strong treatment with MMS (**F**; 6 mM, 45 min), which induced cell death of the whole cell population (insert), was a slight PAR increase noticed with BD anti-PAR. (**G**) WB detecting PAR with BD anti-PAR in control cells or cells exposed to 100 and 250 µM H2O2. (**H**) WB detecting PAR indirectly with the rabbit PAR-binding reagent MABE1031 (1:4000) and anti-rabbit-HRP in control VERO cells or under exposure to 1 mM H2O2 (10 min), 8 mM MMS (45 min), or the PARP inhibitor 3AB (2 h). (**I**) WB detecting PAR with

with respect to the genotoxic treatment. \*: *p* < 0.05, \*\*: *p* < 0.01.

**Figure A5.** γH2AX vs. 53 BP1 foci distribution in one experiment. Foci counting required the following steps, which were done in batch: (1) Separate channels. (2) Segment images to obtain a mask using a fixed threshold for all images of the experiment in each channel. In this case, the following thresholds were used: 50 for the red channel (γH2AX), 62 for the green channel (53BP1), and 40 for the blue (DAPI) channel. (3) The Analyze Particles plug-in allowed for counting objects with certain properties, namely, circularity and size. Foci were defined as ovoid to circular objects (circularity > 0.5) and 5 to 200 pixels. Nuclei were defined as ovoid objects measuring 500 to 500,000 pixels. In each case, the program showed the masks, displayed results, and summarized the data. In this way, we obtained the number of γH2AX foci, 52 BP1 foci, and cell number. (4) In order to determine the **Figure A5.** γH2AX vs. 53 BP1 foci distribution in one experiment. Foci counting required the following steps, which were done in batch: (1) Separate channels. (2) Segment images to obtain a mask using a fixed threshold for all images of the experiment in each channel. In this case, the following thresholds were used: 50 for the red channel (γH2AX), 62 for the green channel (53BP1), and 40 for the blue (DAPI) channel. (3) The Analyze Particles plug-in allowed for counting objects with certain properties, namely, circularity and size. Foci were defined as ovoid to circular objects (circularity > 0.5) and 5 to 200 pixels. Nuclei were defined as ovoid objects measuring 500 to 500,000 pixels. In each case, the program showed the masks, displayed results, and summarized the data. In this way, we obtained the number of γH2AX foci, 52 BP1 foci, and cell number. (4) In order to determine the number of mixed foci (positive both for γH2AX and 53 BP1), mask images (which are 0 or 1 matrixes) were multiplied. In this way, a signal remained in pixels positive for γH2AX and 53BP1 (1 × 1 = 1) while no signal was obtained in a pixel if at least one of the channels was black (1 × 0 = 0). After that, we had objects representing partial foci superimposition. In order to avoid artifacts or overcounting, we used the Analyze Particles Plugin again to count only whole foci. Again, foci were defined as ovoid to circular objects (circularity > 0.5) measuring 5 to 200 pixels. (5) Mixed foci were subtracted from the total γH2AX foci and from the total 53BP1 foci to obtain the numbers of foci with only one mark. (6) γH2AX alone, 53BP1 alone, and mixed foci numbers were normalized by cell number. Data were from a single experiment using 10 microscopic fields (40× zoom 2) per condition. Foci counting involved 247 control cells, 176 BLEO cells, and 119 (BLEO + OLA) cells.
