PARP1 trapping assay

The PARP1 trapping experiment was performed as described before⁴⁹. Briefly, 3000 cells were plated on a poly-l-lysine-coated eight-well chamber slide. After 5 days, cells were either treated with 10 µM olaparib or 10 µM olaparib and 0.2 mM MMS for 4 h. Cells were pre-extracted on ice with 0 PBS supplemented with 0.2% Triton X-100, h 1 µM PARGi (PDD, Fisher, cat. no. 59-521-0) and 10 µM olaparib, for 5 min. Cells were then fixed with 3% PFA, supplemented with 1 µM PARGi and 10 µM olaparib, for 20 min at room temperature. Cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min on ice, then washed with PBS. Blocking was performed for 1 h at room temperature with 0.22 µ-filtered DMEM containing 10% FBS. Cells were incubated with 1:500 dilution of rabbit anti-PARP (Abcam, ab227244) for 1 h at room temperature, followed by washes with 0.02% PBS-Tween. Cells were next incubated with 1:500 dilution of Alexa-Fluor 488 conjugated anti-rabbit secondary (Thermo, A32766) for 1 h at room temperature. Finally, the chamber gasket was removed, and a coverslip was mounted using Prolong Diamond Antifade mounting media with DAPI (Thermo, {"type":"entrez-protein","attrs":{"text":"P36962","term_id":"547832","term_text":"P36962"}}P36962). All images were acquired on a Nikon Ti-2 using a 20x objective.