Olaparib

YHP-836 was synthesized in-house. PARP1/2 inhibitor olaparib was purchased from TargetMol, United States. Temozolomide (TMZ), topotecan, cisplatin, and adriamycin were purchased from J&K Scientific (Beijing, China). Anti-γH2AX and anti-RAD51 were obtained from Cell Signaling Technology (Danvers, MA, United States). An anti-β-actin antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Anti-PARP1 and anti-PARP2 antibodies were from Abcam (Cambridge, United Kingdom). Anti-PAR antibody and HT PARP pharmacodynamic assay kit were purchased from Trevigen (Gaithersburg, MD, United States). The subcellular protein fractionation kit was purchased from Thermo Scientific (Rockford, IL, United States).

Cisplatin, metformin and fenformin were purchased from Sigma-Aldrich (St. Louis, MO, USA); carboplatin from Adipogen (San Diego, CA, USA); paclitaxel from ChemieTek (Indianapolis, IN, USA); doxorubicin and rotenone from Merck; Yondelis (ET-743) from PharmaMar (Madrid, Spain); olaparib from TargetMol (Boston, MA, USA); oxaliplatin, rucaparib, niraparib, KU55933 (ATM inhibitor), AZD6738 (ATR inhibitor), AZD7762 (Chk1 inhibitor) and AZD1775 (Wee1 inhibitor) from Axon Medchem (Groningen, The Netherlands). All the drugs were dissolved in DMSO or water as stock solutions and diluted in medium just before treatment. For cytotoxicity experiments, cells were seeded at 1000–2000 cells/mL and treated with different drug concentrations in 96-well plates 48 h after seeding. After five days of treatment, cell viability was examined with the MTS assay system (Promega Corporation, Madison, WI, USA), and absorbance was acquired using a microplate reader (GloMax Discover, Promega Corporation). Drug concentrations inhibiting growth in 50% of the cells (IC50) were calculated for each cell line, with the interpolation method on Prism 9.5.1 (GraphPad Software, La Jolla, CA, USA). All the experiments were run at least three times in sestuplicate.

The survival was determined by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich/Merck, St. Louis, MO, USA). First, the cells were seeded in 96-well plates at a density of 2 × 10⁴ cells per well. On day two, four, and seven, the cells were treated with Olaparib (TargetMol, Boston, MA, USA), M1, M2, M3, or Cpd1 at 15 different treatment concentrations increasing up to 1024 µM in fresh culture medium each. On day nine, the MTT assay was performed. MTT was first dissolved in 1x PBS (Gibco/ThermoFisher Scientific) to a concentration of 5 mg/mL. Then, the 5 mg/mL MTT solution was diluted in OptiMEM (Gibco/ThermoFisher Scientific) and for HCC-1937 in RPMI without phenol red (Gibco/ThermoFisher Scientific) to a final concentration of 1 mg/mL. An amount of 100 µL of this solution were added to each well. After an incubation time of 2.5 h at 37 °C, the MTT solution was removed and the cells suspended in 200 µL 5% HCl/95% isopropanol (Sigma-Aldrich/Merck). The plates were shaken for 10 min and then the optical density measured at 570 nm with the Tecan Sunrise Photometer (Tecan, Crailsheim, Germany). The MTT experiments were conducted in duplicates and repeated twice each.