Mouse anti parp1

The PLA assays were carried out using a Duolink in situ red starter kit mouse/rabbit kit (Sigma-Aldrich) according to the manufacturer’s protocol. The primary antibodies used were: mouse anti-PARP1 (Sigma-Aldrich, WH0000142M1), rabbit anti-PARP (Cell Signaling), mouse anti-p97 (Abcam, ab11433) and rabbit anti-phospho-H2AX (Cell Signaling). The antibodies were used at a 1:1,500 dilution. Images were acquired on a Marianas advanced spinning disk confocal microscope (3i) and analysed using a custom CellProfiler pipeline. Typically, several hundred nuclei were counted per condition from at least two independent biological repeats.

1 × 10⁵ cells were seeded on glass cover slips prior to treatment in a 24-well plate. After treatment, cells were fixed with 4% paraformaldehyde for 20 min. They were subsequently washed with PBS and blocked with 5% FBS/PBS with 0.3% Triton-X. The cells were incubated overnight at 4 °C with primary antibody (Mouse Anti-PARP1, 1:1000 (Sigma); Rabbit Anti-pH2AX, 1:500 (Cell signalling)) dissolved in blocking solution. After washing with PBS, the cells were incubated for 1 h with secondary antibodies (Goat Anti-Mouse DyLight 488, 1:2000 and Goat Anti-Rabbit Alexa Fluor 568, 1:1000) at room temperature. After the PBS wash steps, the cells were mounted with Roti Mount^® Vectashield containing DAPI and analysed using fluorescence microscopy (Zen Lite, Carl Zeiss, Jena, Germany). Quantification was performed using CellProfiler 3.0 software.