Pdd00017273

Cells were incubated with PARG inhibitor (1 µM PDD 00017273, Sigma) for 10 min and subsequently mock-treated or UV-C irradiated (50 J/m²) and harvested after 10 min. Cell pellets were lysed for 60 min on ice in EBC-1 buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP-40, 2 mM MgCl₂, 1 µM PARP inhibitor Olaparib; 1 µM PARG inhibitor; 1 µM, protease inhibitor cocktail; Roche) supplemented with 500 U/mL Benzonase® Nuclease (Novagen). Cleared lysates were subjected to immunoprecipitation with GFP-Trap®_A beads (Chromotek) for 1.5 h at 4 °C. The beads were then washed with EBC-2 buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, protease inhibitor cocktail; Roche) for six times, EBC-2 buffer supplemented with 300 mM M NaCl (four times) and EBC-2 buffer supplemented with 1 M NaCl (two times). All buffers contained 1 µM PARP inhibitor (Olaparib) and 1 µM PARG inhibitor (PDD 00017273, Sigma). Finally, beads were boiled in Laemmli-SDS sample buffer and immunoprecipitated proteins were resolved by SDS-PAGE and processed for immunoblotting.

Human U2OS osteosarcoma [American Type Culture Collection (ATCC), HTB-96], embryonic kidney 293T (ATCC, CRL-3216), and A549 (ATCC, CCl-185) cell lines were purchased from ATCC. Cells were grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Gibco) and penicillin-streptomycin (100 U/ml; Gibco). All cell lines were cultured in a humidified atmosphere at 37°C with 5% CO₂. Human embryonic kidney 293T (293T) and U2OS cells were plated in 10-cm dishes 24 hours before cells were transfected with the indicated plasmids. 293T cells were transfected using PolyFect (QIAGEN), while U2OS cells were transfected using TransIT-LT1 Transfection Reagent (Mirus Bio), according to the manufacturer’s protocol. Cells were treated with DMSO, 0.5 μM PARP14i (RBN012759, MedChemExpress), 5 μM PARGi (PDD00017273, Sigma-Aldrich), or IFN-γ (100 ng/ml; Merck) for 24 hours.

mESC clone WT #4^{35 (link)} was cultured on tissue culture plates coated with 0.1% Gelatin (Millipore) in 2i medium (Neurobasal—DMEM/F-12 medium (Gibco), supplemented with 1x N2 (Gibco), 1x B27 (Gibco), 2 mM L-Glutamine (Gibco), 1000 U/ml Leukemia inhibitory factor (LIF, Millipore), 100 U/ml PEN-STREP (Gibco), 1 µM PD0325901 (Sigma), 3 µM CHIR99021 (Sigma), 50 µg/ml BSA) at 37 °C in 5% CO₂ and 20% O₂. HEK293T (ATCC number CRL-11268) and DIvA cells^{24 (link)} (kind gift from G. Legube) were cultured in DMEM (Gibco) supplemented with 10% FBS Gold (PAA), 2 mM L-Glutamine, and 100 U/ml PEN-STREP at 37 °C in 5% CO₂ and 20% O₂. All cell lines were tested negative for mycoplasma contamination.
Cells were transiently transfected with siRNAs with Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. siRNA knockdown efficiencies were routinely assessed by reverse transcription (RT)-coupled qPCR, and were at least 60% efficient. For PARG inhibition, mESCs were treated with 0.1–10 µM PDD00017273 (Sigma) for 48 h. PARP inhibititon was for 24 h with 1 µM olaparib (Selleckchem, S1060).