Parg inhibitor

U2OS 2-6-3 cells containing 200 copies of a LacO-containing cassette were plated on an 18-mm glass coverslip. The next day the cells were co-transfected with lipofectamine 2000 (Invitrogen) and plasmid DNA for 6 h at 37 °C. Next the medium was replaced with DMEM +/+ and incubated overnight at 37 °C. Prior to the UV-C micro-irradiation, the medium was replaced with CO₂-independent Leibovitz L15 medium (Thermo Fisher Scientific) and cells were incubated with 10 µM of PARG inhibitor (Sigma) for 30 min. If indicated the cells were additionally incubated with 10 µM Olaparib. UV-C laser tracks were made using a diode-pumped solid-state 266-nm Yttrium Aluminum Garnet laser (average power 5 mW, repetition rate up to 10 kHz, and pulse length 1 ns). The UV-C laser is integrated into a UGA-42-Caliburn/2 L Spot Illumination system (Rapp OptoElectronic). Micro-irradiation was combined with live-cell imaging in an environmental chamber set to 37 °C on an all-quartz widefield fluorescence Zeiss Axio Observer 7 microscope, using a ×100 (1.2 NA) ultrafluar glycerol-immersion objective (UV-C). The laser system is coupled to the microscope via a TriggerBox, and a neutral density (ND-1) filter blocks 90% of the laser light. An HXP 120-V metal-halide lamp was used for excitation. Images were acquired in Zeiss ZEN and quantified in ImageJ.

Cells were treated with 200 μM H₂O₂ for 20 min, then cells were scraped and washed once in phosphate-buffered saline (PBS), before immediate lysis in either 4× SDS loading dye (diluted to 1X in lysis buffer following cell lysis) or lysis buffer containing PARG inhibitor (20 mM Hepes pH7.5, 250 mM KCl, 5% glycerol, 10 mM MgCl₂, 0.5% Triton X-100, 1 μΜ PARG inhibitor (PDD00017273, Sigma), to preserve PAR modifications. Lysates were sonicated before being immunoblotted as before.

Cells were harvested by trypsinization and lysed in TEB250 lysis buffer (50 mM HEPES pH 7.4, 250 mM NaCl, 2 mM MgCl₂, 5 mM EGTA pH 8, 1 mM DTT, 0.5% Triton X-100, 10% glycerol, protease inhibitor cocktail (Roche), phosphatase inhibitors cocktail (PhosSTOP, Sigma), 50 μM PARPi, 100 μM PARG inhibitor and benzonase (Sigma; 1:1,000)) for 45 min on ice, followed by centrifugation (15 min at 16,000g). For immunoprecipitation, 0.5 mg or more of the protein lysate was incubated with antibody to mono/poly-ADP-ribose antibody (Cell Signalling; Supplementary Table 1) for 2 h and then with Protein G Dynabeads (Invitrogen) for 2 h. The beads were then washed (3×) with lysis buffer, resuspended in sample buffer and heated for 5 min at 90 °C and subjected to SDS–PAGE. The proteins were transferred to nitrocellulose and immunoblotted with ADP-ribose-binding reagent (Millipore; Supplementary Table 1).