Olaparib

Antibodies we used are as follows: Secondary antibodies: goat anti‐rabbit (ZB-2301) and rabbit anti‐mouse (ZB-2305) secondary antibodies, Alexa Fluor 594-labeled goat anti-mouse (ZF-0513) and Alexa Fluor 488-labeled goat anti-rabbit antibodies (ZF-0511) were purchased from ZSGB-BIO. Anti-RAD51 antibody (Abcam; ab133534), FOXM1 (D12D5) XP® Rabbit mAb (Cell Signaling; #5436), Anti-gammaH2A.X (phospho S139) antibody (Abcam; ab26350), Mouse Anti-βactin mAb (ZSGB-BIO; TA-09).
Pharmacological agents: Olaparib (MedChemExpress; HY-10162), DMSO (Solarbio; D8371).
Plasmid DNA was isolated by plasmid DNA extraction kits (CWBIO; CW2105S) using the alkaline lysis method.

Recombinant Human TNF-α (Cat. No. 300-01A), IFN-γ (Cat. No. 300-02), and insulin growth factor (IGF; Cat. No. 100-11) were purchased from PeproTech (Cranbury, NJ, USA). Sodium butyrate (Cat. No. S1200) and cisplatin (Cat. No. C2210000) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The inhibitors CUCD-101 (Cat. No. HY-10223), decitabine (HY-A0004), santacruzmate A (HY-N0931), zebularine (HY-13420), olaparib (HY-10162), alpelisib (HY-15244), and MK-2206 (HY-108232) were all purchased from MedChemExpress (Sollentuna, Sweden). Fulvestrant (ICI 182,780; Cat. No. 1047) was obtained from Tocris Bioscience (Bristol, UK). A medium containing 0.5% DMSO was used as a vehicle-only control for the experiments.

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.

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.

Epithelial ovarian cancer (EOC) cell lines PEO1 (BRCA2^−/−) were kindly provided by Dr. Rugang Zhang (Wistar Institute), Kuramochi (BRCA2^+/−) and OVCAR4 (BRCA-WT) cells were kindly provided by Dr. Adam Karpf (Eppley Institute, University of Nebraska), UWB1.289 (BRCA1^−/−) and OVCAR3 (BRCA-WT) cells were purchased from ATCC (Manassas, VA). UWB1.289 cells were cultured in a 1:1 mixed medium solution containing 50% RPMI-1640 medium and 50% MEGM with bullet kit (Lonza, MEGM bullet kit, CC-3150) supplemented with 3% fetal bovine serum (FBS). Other cells were cultured in RPMI-1640 media supplemented with 10% FBS. 100 μg/ml streptomycin and 100 units/ml of penicillin solution were added to the media. Cells were cultured at 37 °C in a humidifying chamber with 5% CO₂. All the experiments were performed using cells within 20 passages after being revived from liquid nitrogen. All-trans-Retinoic Acid (ATRA) was purchased from Sigma Aldrich (Cat. No. R2625), RAR agonist CH55 (Cat. No. 2020) and RAR antagonist AGN 193109 (Cat. No. 5758) were purchased from Tocris Biosciences. Olaparib was purchased from MedChemExpress (Cat. No. HY-10162). The ALDH1A1 inhibitor NCT-505 was synthesized by the National Center for Advancing Translational Sciences (NCATS). All the above-mentioned drugs were dissolved using DMSO and fresh stock solutions were made each time before use.

Olaparib (HY-10162) was purchased from MedChemExpress (MCE) and prepared in dimethyl sulfoxide (DMSO) (Sigma, D2660) at 100 mmol/L and stored in aliquots in −80 °C. Carboplatin (HY-17393) was obtained from MCE and prepared in ultrapure water at 10 mmol/L and stored in aliquots in −80 °C. Ai-Ling#1 (ATO) solution (1 mg/ml) was obtained from Harbin Yida Pharmaceutical Co. Ferroptosis inhibitors Ferrostatin-1 (Fer-1, HY-100579), Liproxstatin-1 (Lip-1, HY-12726), Deferoxamine mesylate (DFO, HY-B0988), apoptosis inhibitor Z-VAD-FMK (HY-16658B), and oleic acid (OA, HY-N1446) were purchased from MCE. Compound C (CC, S7840) was purchased from Selleck Chemicals.