Niraparib

The experimental setup has been described previously^{11 (link)}. In brief, 24 h after seeding, cells were treated with medium and antibiotics containing 0.1, 0.5, 1, 5, 10, 25, or 50 µM Niraparib (Selleckchem, Houston, TX), 0.1, 0.5, 1, 5, 10, 50, 100, or 200 µM Olaparib (Selleckchem, Houston, TX), 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, or 1 µM Berzosertib (Axon Medchem, Groningen, NL), or a combination of the respective concentrations of Berzosertib and Niraparib (all final well concentrations) and were incubated at 37 °C with 5% CO₂ for six days. Medium containing the respective drug concentrations was replaced every 48 h. On day 6, 100 µM 5′-bromo-2′desoxyuridine (BrdU) labeling solution was added and the rate of proliferating cells was determined 24 h later (=d7) by means of a BrdU-ELISA (Roche, Basel, SUI), according to the manufacturer’s instructions. Optical density was measured at a wavelength of 370 nm and a reference wavelength of 492 nm using a microplate reader (Tecan Spark, Männedorf, SUI).

Olaparib (Catalog# A4154), cisplatin (Catalog# A8321), carboplatin (Catalog# A2171), and 5-fluorouracil (Catalog# A4071) were all purchased from APExBIO company. UPF 1096 (Catalog# S8038), NMS-P118(Catalog# S8363), stenoparib (E7449) (Catalog# S8419), niraparib (Catalog# S2741), rucaparib (Catalog# S4948), and veliparib (ABT-888) (Catalog# S1004) were all purchased from Selleckchem.
cisplatin (Catalog# A10221) was purchased from AdooQ Bioscience. ART-558(Catalog# HY-141520), DNA-PK inhibitors PIK-75 hydrochloride (Catalog# HY-13281), Nedisertib (Catalog# HY-101570) and AZD-7648 (Catalog# HY-111783), RAD51 Inhibitor B02 (Catalog# HY-101462), and Olaparib (for in vivo experiment: Catalog# HY-10162) were all purchased from MCE (MedChem Express). ART-812 was synthetized by Dr. Wayne Childers at Temple University School of Pharmacy. All compounds were dissolved, aliquoted, and stored following the manufacturer’s instructions.

Olaparib, veliparib, and niraparib were obtained from Selleck Chemicals and cisplatin was obtained from Biovision. To test cellular viability after Olaparib treatment, cells were seeded in 96-well plates at a density of 2000 cells per well, treated with the indicated doses of Olaparib for 3 days, and assessed using CellTiter-Glo reagent (Promega, G7572) per manufacturer’s instructions. Clonogenic survival assays were performed by seeding cells in 6-well plates; after 72 h (Olaparib) or 24 h (cisplatin) of treatment at the indicated concentrations, media was changed and colonies were allowed to form for 2 weeks. Cells were fixed with a solution of 10% methanol + 10% acetic acid and stained using crystal violet (2% solution, Aqua Solutions). For apoptosis assays, cells were treated with Olaparib (5 μM) for 3 days, prepared for flow cytometry using the FITC Annexin V kit (Biolegend, 640906) and quantified using a BD FACSCanto 10 flow cytometer operated by BD FACSDiva 8.0.1 software.

To determine colony formation ability, 300–500 established CCA cells were plated in each well of a 6-well plate as described previously [41 (link)]. Drugs were added 24 h later and cells were allowed to grow until colonies appeared. Crystal violet (Sigma-Aldrich, St. Louis, MO, USA) was used to stain the colonies. The number of colonies was counted and the data were presented as a bar graph. For spheroid formation in PDXC, 1000 cells per well were plated, treated with the indicated drugs and allowed to grow for 6 days. Niraparib, olaparib and gemcitabine were purchased from Selleck chemicals (Houston, TX, USA).

Olaparib was purchased from Cambridge Biosciences (UK) and veliparib, talazoparib and niraparib (MK4827) were purchased from Selleckchem (UK). All were dissolved in 100% DMSO to give a 10 mM stock. When confirming inhibition of PARP, PARP inhibitors were added overnight (16 h) for convenience. Temozolomide and camptothecin were purchased from Sigma-Aldrich (UK).