Rucaparib

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.

Both human CAL27 and SCC25 cell lines were obtained from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37° C in a 5% CO2 humidified incubator. The medium was changed every 2 days. Cells in the mid-log phase were used in the experiment. Oxaliplatin (ab141054) was purchased from Abcam (MA, USA). N-Acetyl Cysteine (NAC, 3 mM) and rucaparib (1mM) were purchased from MedChemExpress (MCE, NJ, USA).

Olaparib (HY-10162), rucaparib (HY-10617A), and sunitinib (HY-10255A) were purchased from MedChemExpress. 5F02 was obtained from the ChemDiv Representative Diversity Set, comprising 50,000 molecules. Samples were validated by ChemDiv1H-NMR and HPLC/LCMS for ≥95% purity. For cell culture studies, inhibitors were dissolved in DMSO at 10 mM stock concentration and kept at −80 °C protected from light. Final dilutions were made in culture media. For the in vivo studies, drug compaunds were diluted from powder in 10% 2-hydroxypropyl-β-cyclodextrin on PBS and used freshly made for each injection. Doxycycline (Millipore-Sigma, Darmstadt, Germany, #9891) and puromycin (Clontech/Takara, Mountain View, CA, USA, 631,306) were dissolved in DMSO at stock concetrations of 100 ug/mL and 1 mg/mL, respectilvely, and kept at −20 °C.

UM-UC-3, T-24 (human bladder cancer cell lines), and SV-HUC-1 (normal human bladder epithelial cell line) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured in EMEM, McCoy’s 5a, or F12K media respectively, supplemented with 10% FBS and penicillin/streptomycin. Cells were used within half a year after being received from ATCC or after thawing from cryopreservation. Short Tandem Repeat (STR) profiling is used by the ATCC for cell line authentication. The cell lines in culture were routinely tested for mycoplasma contamination every two months using the MycoFluor^TM Mycoplasma detection kit (Thermo Fisher Scientific, Waltham, MA). Tubulin antibodies were obtained from Thermo Fisher Scientific, Waltham, MA. Cleaved and whole caspases 3, 7, and 9 antibodies were obtained from Cell Signaling Technology, Danvers, MA. Ki-67 antibodies were obtained from Neomarker, Fremont, CA. The PARP inhibitors, olaparib, niraparib, rucaparib, veliparib, and talazoparib, were obtained from MedChem Express, Monmouth Junction, NJ. Cisplatin was from Sigma Aldrich, St. Louis, MO. Other reagents were supplied by local suppliers such as Fisher Scientific and VWR International.