*4.2. Treatments with BLEO and Inhibitors*

Bleomycin sulfate treatment (BLEO); from NOLVER (Montevideo, Uruguay), NIPPON KAYAKU (Japan), or LKM (Peru) consisted of a 45 min pulse in the absence of FBS. Controls and other experimental conditions were also subjected to 45 min FBS depletion. Initially, dose–response curves (0, 4, 10, 20, 40, 80, 160, 200, and 500 µg/mL) were found. The Olaparib (Tocris, Minneapolis, MN, USA) treatment was continuous. Dose–response curves (0, 50, 100, 150, and 200 nM) were also found. Then, 40 µg/mL BLEO and 50 nM OLA were selected for the following experiments: OLA effect on the PAR pool was checked and compared with other PARPis and with the PARG inhibitor DEA in untreated VERO cells using ICF with a BD anti-PAR antibody (Figure A3B). While DEA increased PAR, 25 nM OLA did induce an effect that was very similar to 250 nM OLA, diminishing the endogenous PAR by more than 100 nM EB or 5 mM 3AB. Like with OLA, the treatment with ATM, DNA-PK, and Lig IV inhibitors, namely, 10 µM KU55933 (SIGMA SML-1109, St. Louis, Missouri, USA [77]), 0.1 µM KU-0060648 (SIGMA SML-1257, St. Louis, MO, USA), and 0.1 µM SCR7 Pyrazine (SIGMA SML-1546, St. Louis, MO, USA [78]), was continuous. SIGMA is subsidiary of Merck KGaA (Darmstadt, Germany), and its headquarter is at St. Louis, Missouri, USA.Co-treatments were done with the correspondent inhibitors + BLEO for 45 min; then, for the MTT or clonogenic experiments, BLEO was removed and the inhibitors were added again in a fresh medium with FBS. For the comet assay or ICF, the experiment was stopped and the cells were fixed or lysed immediately after the 45 min treatment. All times in the graphs refer to the post-BLEO-treatment time. As such, t = "0" means "immediately after the 45 min BLEO treatment", t = 24 is 24 h later, and so on.
