Pdd00017273

COS-7 cells were co-transfected with a mixture of GFP, GFP-SET8 FL or GFP-SET8, 3xFLAG or 3xFLAG-PARP1, HA-ubiquitin plasmids, and Fugene HD transfection reagent (Promega, # E2311) according to manufacturer’s recommendations. After 48 h, transfected COS-7 cells were treated or not with 50 μM MG132 for 2 h and lysed as described previously^{60 (link)}. Synchronized GFP-SET8 FL transfected cells were treated for 3 h with 10 μM cullin inhibitor (MLN4924, Selleckem # S7109), PARG inhibitor (PDD 00017273, Tocris # 5952) or with DMSO as control. Cell lysates (100–200 μg) were then immunoprecipitated with GFP antibody (Thermo Fisher Scientific # G10362). Ubiquitination and ADP-ribosylation were detected by Western blot using HA-tag antibody (Cell signaling Technology # 3724S) or anti-ADPribose antibody, respectively.

HeLa cells were provided by Riken (Tsukuba, Japan) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml), in a humidified incubator with 5% CO₂ at 37 °C. 3-Aminobenzamide (3AB) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Olaparib was purchased from ChemScene (NJ, USA), emetine hydrochloride from Cayman Chemical (No. 21048; MI, USA), and PDD00017273 [37 (link)] from Tocris Bioscience (MN, USA), and PD0325901 from ChemScene. The following antibodies were obtained from Santa Cruz Biotechnology (TX, USA): mouse anti-human PARP1 IgG (sc-8007), mouse anti-human phosphorylated ERK IgG (sc-7383), mouse anti-human ERK2 IgG (sc-1647), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (sc-2005), and HRP-conjugated goat anti-rabbit IgG (sc-2004). Rabbit anti-human PARG IgG (ab16060) was purchased from Abcam (Cambridge, UK). Anti-PAR (10H, IgG3 kappa), secreted from hybridoma cells [38 (link)], was purified using a Protein A Sepharose Fast Flow column (GE Healthcare, IL, USA). Anti-PAR polyclonal antibodies were produced in rabbits by injecting PAR mixed with methylated BSA in our laboratory [39 (link),40 ]; the antibodies were purified with a Protein A Sepharose Fast Flow column.

Human MDA-MB-231, MCF7, and HeLa cell lines were obtained from the European Collection of Authenticated Cell Cultures and BT-20 and MCF 10A cell lines from the American Type Culture Collection. Cells were maintained in culture for less than 20 passages after receipt under standard conditions (37°C, 5% CO₂) and were regularly tested for mycoplasma contamination. All cell lines except MCF 10A cells were maintained in Dulbecco’s Modified Eagle Medium from Gibco, supplemented with 10% fetal bovine serum and antibiotics. MCF 10A cells were cultured using the MEGM^™ Mammary Epithelial Cell Growth Medium BulletKit^™ (Lonza). Cells were treated with the following drugs: veliparib (ABT-888; Enzo Life Sciences), olaparib (AZD2281; MedChemExpress), a PARG inhibitor (PDD 00017273; Tocris, bio-techne), H₂O₂ (Sigma-Aldrich), rapamycin (Sigma-Aldrich), triptolide (Sigma-Aldrich), lactacystin (Sigma-Aldrich).

The following small molecule inhibitors were used in this study: PARPi (Olaparib; Selleck Chemicals, catalog no. S1060), PARGi (PDD 00017273; Tocris Bioscience, catalog no. 5952), ATRi (AZD6738; Selleck Chemicals, catalog no. S7693) and FEN1i (MedChem Express; catalog no. HY-136485).

All NSC compounds were obtained from the Developmental Therapeutics Program (DTP) repository at NCI/NIH. JA1-5 (JS-2088), JA5-8 (BAS 05169959), and JA5-9 (AO-476/12797006) were purchased from Ryan Scientific. JA2-9 (Z57032584), JA5-10 (EN300-63858), and JA5-11 (Z385453050) were purchased from Enamine. JA2-8 (JFD03560SC) was purchased from Maybridge, and JA5-7 (F3350-0573) was purchased from Life chemicals. All inhibitors were dissolved at 40 mM concentrations in DMSO. Hydroxyurea, Nedaplatin, Doxorubicin, Veliparib, and Olaparib were purchased from SelleckChem. PDD00017273 was from Tocris.

PARP inhibitors olaparib (S1060; Selleck Chem) and talazoparib (S7048; Selleck Chem) and PARG inhibitor PDD00017273 (5952; Tocris Bioscience) were used at a final concentration of 5 µM, 50 nM and 0.3 µM, respectively, and were added to the cells 1 h prior to and during micro‐irradiation. Transcription inhibitors were employed as follows: α‐amanitin (20 µg/ml) for 8 h; DRB (100 µM) for 2 h; and actinomycin D (5 nM and 2.5 µM) for 40 min prior to and for the entire duration of micro‐irradiation. For RNase treatment, cells were first washed with warm PBS and permeabilized with Tween‐20 (1% (v/v)) in PBS for 5 min followed by treatment with 1 mg/ml RNase A (Thermo) for 10 min at room temperature (RT). Following the respective treatments, cells were micro‐irradiated and imaged immediately.