Parp1

Cell lysates were obtained by incubating a cell pellet with RIPA buffer for 30 minutes. Lysates were than resolved by SDS-PAGE. The proteins were transferred onto an Immobilon-P PVDF membrane (Millipore), which were blotted overnight with primary antibodies recognizing GAPDH, DNA LIG4 (Santa Cruz Biotechnologies), Ku70, RAD51 or PARP1 (ThermoFisher Scientific). This was followed by 1 h incubation with secondary antibodies conjugated with HRP (Anti-Mouse and Anti-Rabbit antibodies, Cell Signaling).

The DuoLink (Merck) PLA assay was performed according to the manufacturer's protocols. Cells were seeded in 12 mm glass coverslips 48 h before the experiment and treated if needed, fixed with 4% PFA at RT for 10 min, and permeabilized for 10 min at RT in PBS supplemented with 0.1% Triton X‐100 (Sigma Aldrich). For blocking, cells were incubated for 1 h at 37°C in the provided blocking solution. Incubation with primary antibodies (PARP1, Thermo Fisher, Cat#MA 3‐950, TSG101, Thermo Fisher, Cat#MA 1‐23296, both diluted 1:200) was done overnight at 4°C.

In a reaction volume of 10 μl, 1 or 10 pmol ALC1^fl or ALC1^cat were incubated with 0.2 pmol Cy3/Cy5-labeled mononucleosomes for 30 min at 32°C in buffer containing 20 mM HEPES, pH 7.9, 50 mM NaCl, 4.5 mM MgCl₂, 10% glycerol, 0.02% Triton X-100, 2 mM DTT, 45 μg/mL BSA, and 2 mM ATP. Where indicated, reactions contained 1 pmol PARP1 (Thermo Fisher) pre-incubated with 50 μM NAD⁺ for 5 min at 37°C. Reaction products were incubated with 10 U HhaI for 30 min at 37°C. Histone proteins were degraded by incubation with 1 mg/mL Proteinase K for 1 h at 37°C and the samples were analyzed on a 15% polyacrylamide/8 M urea gel that was imaged for Cy5.

To analyze the protein expression of EMT markers and PARP-1 was performed the immunofluorescence assay according to Gelaleti et al. (2017) [17 (link)]. Briefly, 6 × 10⁴ cells of CF41 and MDA-MB-468 cell lines were transferred to a silicone separator attached to a slide and kept in the incubator for 24 h at 37 °C with 5% CO₂. Then, the cells were treated with carboplatin, losartan, or both in combination. For the immunofluorescence technique were used the specifics primary antibodies E-cadherin (1:200) (catalogue number 3195S, Cell Signaling Technology, Danvers, MA, USA), N-cadherin (1:100) (catalogue 7939, Santa Cruz Biotechnology, Dallas, TX, USA) and PARP-1 (1:50) (catalogue 436400, Thermo Fisher Scientific, Waltham, MA, USA) and the secondary antibody used was Alexa Fluor 648 anti-mouse IgG (1:100) (Sigma-Aldrich). To analyze the protein expression, the cells were captured using a microscope containing a specific software (OLYMPUS, model BX53, software Image-Pro Plus version 7.0, Rockville, MD, USA). All experiments were carried out in triplicate and protein expression was quantified according to Jardim-Perassi et al. (2014) [18 (link)] as cited by Gelaleti et al. (2017) [17 (link)]. The analyses were performed using ImageJ Software (NIH, Bethesda, MD, USA) and all values were obtained in arbitrary units (a.u.) and represented as the mean optical density (M.O.D.).

ATPase activity was measured as described previously (Ahel et al., 2009 (link)) by monitoring the fluorescence intensity of a phosphate binding protein (Brune et al., 1994 (link), Ferreira et al., 2007 (link)) labeled with a coumarin-based fluorescent dye, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin (MDCC), as a readout of the amount of inorganic phosphate (P_i) generated by ATP hydrolysis. Upon binding P_i, the fluorescence of the labeled phosphate binding protein (MDCC-PBP) increases and its emission wavelength shifts. The increase in fluorescence was recorded in solution using a Spark 10 M (Tecan) instrument. 25 μL ATPase reactions were carried out in 50 mM Tris pH 7.9, 50 mM NaCl, and 5 mM MgCl₂ buffer with 80 nM ALC1 and 1 mM ATP. Where indicated, 50 μM NAD⁺ and 80 nM PARP1 (Thermo Fisher) and/or 120 nM 239 bp dsDNA were added. Upon incubation for 2 h at 37°C, phosphate sensor (Thermo Fisher) was added at a final concentration of 0.5 μM and immediately measured (excitation = 420 nm, emission = 465 nm).

The cells were plated (6 × 10⁴ cells) and submitted to gene edition of AGTR-1 by CRISPR/Cas9. After, cells were fixed in 4.0% paraformaldehyde and the primary antibodies were used to delineate the expression of corresponding antigens: VEGF (1:300) (Santa Cruz Biotechnology, Dallas, TX, USA) and PARP-1 (1:50) (catalogue 436400, Thermo Fisher Scientific) The immunocytochemistry staining procedures were performed using Reveal System of Detection without Biotin Kit (Biogen, Cambridge, MA, USA) according to the manufacturer’s instructions. Briefly, cells were washed with distilled water and incubated with citrate buffer at 96 °C for 35 min for antigen retrieval. Once it was washed with PBS the sections were incubated with 0.1% of hydrogen peroxide for 15 min and protein blocking for 10 min. Following this process, sections were incubated with the primary antibody diluted in PBS/albumin 5% at 4 °C overnight and with secondary antibody (anti-mouse and -rabbit immunoglobulins). The sections were washed once again and incubated with HRP peroxidase conjugate followed by chromogenic substrate (3,3′-diaminobenzidine tetrahydrochloride—DAB) (Biogen, Cambridge, MA, USA). At the end, all sections were counter stained with hematoxylin, dehydrated and cover slipped with Erv Mount (Easypath, Sao Paulo, Brazil).