Niraparib

Vorinostat was obtained from LC Laboratories (Boston, MA), olaparib and niraparib were obtained from MedChemExpress (Monmouth Junction, NJ), salubrinal was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), raphin-1 was obtained from Tocris Bioscience (Bristol, UK), and veliparib was obtained from APExBIO (Houston, TX). All the drugs were added from stock solutions in dimethyl sulfoxide (DMSO), and the control cultures received equal amounts of the vehicle. The final concentration of DMSO in the culture medium did not exceed 0.1%. All the other materials were of analytical grade.

The compounds used in this study include LCL161 targeting XIAP, Fludarabine inhibiting DNA synthesis, OSI‐027 blocking mTOR, Rucaparib, and Niraparib targeting PARP1, which were chosen based on their selective inhibitory effect on MDA‐MB‐231 cells (Garnett et al,2012; Iorio et al,2016). Compounds LCL161, Fludarabine, OSI‐027, Niraparib, and Rucaparib were obtained from MedChemExpress and dissolved in DMSO. For scRNA‐seq experiment, 0.1 µM of LCL161, 0.15 µM of Fludarabine, 2.5 µM of OSI‐027, 15 µM of Rucaparib, and 12.5 µM of Niraparib were used to treat the cells for 4, 8 or 24 h.

Human RCC cell lines Caki-2 (representing ccRCC), ACHN (representing pRCC) and the normal human renal epithelial cell line RPTEC/TERT1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and cultured in McCoy’s 5a modified medium with 10% complete FBS; Eagle’s Minimum Essential Medium with 15% FBS; and DMEM: F12 medium supplemented with hTERT RPTEC Growth kit as recommended by ATCC, respectively. All experiments with cell lines were performed either within 6 months of receipt from ATCC or resuscitation after cryopreservation. ATCC uses Short Tandem Repeat (STR) profiling for testing and authentication of cell lines. Antibodies against Tubulin were from Thermo Fisher Scientific. Antibodies against cleaved caspase-3, cleaved caspase-7, cleaved caspase 9, cleaved PARP, whole caspase 3, whole caspase 7, whole caspase 9, and whole PARP were from Cell Signaling Technologies. PARP inhibitors (niraparib, olaparib, rucaparib, talazoparib, and veliparib) were obtained from MedChem Express. All other reagents were of analytical grade and obtained from local suppliers.

In vitro PARylation was achieved by incubating 5 μM purified recombinant murine TET1-CD or TET2-CD with 0.18 μg of commercial human recombinant PARP-1 or PARP-2 (Enzo Life Sciences, Inc.) in 50 mM Tris–HCl pH 8, 1.5 mM DTT, 1 mM MgCl₂, 0.25 μg/μl DNK salmon sperm, 200 μM NAD⁺ at room temperature (RT). The reaction was stopped after 5 min, 15 min, 30 min or 60 min by adding sample buffer. In the case of TET2 in vitro PARylation, one of each PARP-1 and PARP-2 reactions were stopped after 60 min by 8 μM niraparib (MedChemExpress) and then PARG enzyme (0.043 ng/ml) (Trevigen) was added and incubated at 37 °C for 1 h.
All samples were separated by Tris–glycine SDS-PAGE on 10% polyacrylamide gels and electro-transferred onto nitrocellulose membranes. Membranes were stained by Ponceau-S, photographed and probed by primary murine anti-PAR (1:1000, Enzo Life Sciences, Inc., H10) or a murine anti-HIS (1:2000, Roche) antibody and secondary anti-mouse horseradish peroxidase-conjugated secondary antibody (1:10,000, GE Health care). Signal was visualised on X-ray film with the enhanced chemiluminescence solution reagent (Thermo Scientific).