Azd2281

PARPi (O, olaparib, AZD2281), ATRi (A, ceralasertib, AZD6738), and CHK1i (C, MK-8776) were purchased from Selleck Chemicals (Houston, TX, USA). The stock solutions of studied inhibitors were separately prepared from powders dissolved in 100% dimethyl sulfoxide (DMSO), aliquoted, and stored at −80 °C for up to a maximum of six months. RPMI 1640 and DMEM culture media, heat-inactivated fetal bovine serum (HI-FBS), and trypsin-EDTA were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Details concerning other key reagents used in the studies are included in Section 2 and Supplementary Tables (Tables S1–S3). Chemicals and solvents were obtained from Sigma-Aldrich (Saint Louis, MO, USA) or Avantor Performance Materials Poland S.A. (Gliwice, Poland).

U2OS cells were plated on to 10 cm dishes at 1 × 10⁶/dish. The next day cells were transfected with 40nM siRNA as detailed above. 24 h post transfection the cells were replated to a 96-well plate at 6 × 10⁴/well and were left O/N in 5% CO₂ incubator at 37°C. The next day cells were treated with 5-hmdU, PARGi, and/or PARPi (Olaparib, AZD2281, SelleckChem) in full DMEM, O/N in 5% CO₂ incubator at 37°C. The next day the full DMEM was removed, and the cells were washed with unbuffered DMEM and incubated with 5-hmdU, PARGi, and/or PARPi in unbuffered DMEM in 0% CO₂ incubator at 37°C for 1 hr. Basal oxygen consumption rate (OCR) and basal extracellular acidification rate (ECAR) were measured simultaneously (8 replicate wells) in real time. Oligomycin (1 μM) at injection port A, FCCP (300 nM) at injection port B, 2-DG (100 mM) at injection port C, and rotenone (1 μM) at injection port D were injected to change bioenergetic compacity and automatic measurements were captured. The basal OCR and ECAR was reported as pmol/min and mpH/min, respectively. Statistical significance was assessed through a one-way ANOVA using GraphPad Prism.

A549 and HeLa cells (5×10⁴) were seeded into 25 cm² flasks in 2 ml of growth medium, Medium was replaced with fresh growth medium with or without IFN gamma (25 ng/ml) 16-24 h after seeding. Twenty-four or 48 h after addition of IFN gamma, medium was replaced with fresh medium containing olaparib (1, 1.5 or 5 microM)(AZD2281, Selleckchem, Houston, TX) or cisplatin (2.3, 4, or 8 microM)(Sigma-Aldrich). Three days after addition of olaparib or cisplatin, cells were washed to remove the dead cells and particles and adherent cells were trypsinized and enumerated using a Coulter counter (Beckman, Mississauga, ON). Viability of the counted cells was confirmed by Trypan blue exclusion. H441 cells (5×10⁴) were seeded into 6-well plates in 3 ml of growth medium. Medium was replaced with fresh growth medium containing cisplatin (5 or 10 microM) 16-24 h after seeding. Seven days after addition of cisplatin, cells were washed to remove the dead cells and particles and adherent cells were trypsinized and enumerated by Coulter counting.

U2-OS osteosarcoma cells were cultured in McCoy’s 5a modified medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FCS; Capricorn Scientific and PAA Laboratories), 100 U/ml penicillin and 100 μg/ml streptomycin (both Thermo Fisher Scientific). U2-OS control and shDEK cells [44 (link)] were additionally supplemented with 2 μg/ml puromycin (Merck). puromycin was omitted 36 h prior to experiments. U2-OS shDEK cells stably express an shRNA targeting the human DEK transcript, resulting in a permanent reduction of DEK protein levels of around 90% [44 (link)]. U2-OS wildtype cells were a kind gift of G. Marra, University of Zurich, Switzerland. HeLa S3 cervical adenocarcinoma cells were cultured in DMEM medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 6 mM L-glutamine (Thermo Fisher Scientific). BJ-5ta foreskin fibroblasts were cultured in a 4:1 mixture of DMEM medium and Medium 199 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, 4 mM L-glutamine and 10 μg/ml hygromycin B (Merck).
For induction of replication stress, cells were treated with hydroxyurea (HU; Merck) or camptothecin (CPT; Merck) as indicated. PARP1/2 activity was inhibited with ABT-888 or AZD-2281 (both Selleckchem) as indicated.