Ve 821

Short-term (72 h drug exposure) was performed as previously described^{21 (link)}.
For long-term exposure, cells were seeded at low density in a six-well plate. Treatment started 24 h later, and refreshed twice weekly until 80% confluency. Cells were stained using crystal violet (Sigma-Aldrich) after fixation using 2% paraformaldehyde (Sigma-Aldrich, Zwijndrecht, The Netherlands).
KU-60019, Wortmannin, ETP-46464, VE-821, MK-8776 (SCH 900776), PF-477736, LY2603618/Rabusertib, and Palbociclib were purchased from Selleckchem (Munich, Germany), LY2606368/Prexasertib from Medchem (Sollentuna, Sweden). Adavosertib (MK-1775) from Biovision (Milpitas, USA). All were dissolved in a DMSO stock dilution (10 mM), except Palbociclib (10 mM in ddH₂O). Assays contained <1% DMSO. All drug-survival assays depict the standard error of the mean (SEM) of three independent experiments in triplicate.

The following antibodies were used: γH2AX (Active Motif, 39117), H3 (Active Motif, 31277), CHK1 (Santa Cruz, sc-8408), CHK1pS317 (Cell Signaling Technology, 2344P), Actin (Sigma, A5441), BrdU (BD Biosciences, 347,580). Secondary antibodies were as follows: horseradish peroxidase-conjugated anti-mouse IgG (BD Biosciences, 554002) and anti-rabbit IgG (Vector Laboratories, PI-1000) for western blotting. Drugs used in this study include: HU (ThermoFisher Scientific), APH (Sigma), MMS (ThermoFisher Scientific), and VE-821 (Selleckchem).

For knockdown experiments, cells were transfected 24–96 h before sample collection with the indicated siRNA using RNAiMax transfection reagent (Life Technologies) according to the manufacturer's instructions: siLUC (100–200 nM; 5′-GGUACGCGGAAUACUUCGAdTdT-3′), siFZR1, siRad51, siAPE1, siTDG (100–200 nM siGENOME SMARTpool Dharmacon). The following reagents were used to treat ES cells for the indicated time at the indicated final concentrations before collection: ATM inhibitor (KU55933, Kudos; 8 h, 10 μM); ATR inhibitor (ETP-46464, provided by O. Fernandez-Capetillo, CNIO, Madrid; 8 h, 5 μM; VE-821, Selleckchem; 8 h, 10 μM); PARP inhibitor (Olaparib, Selleckchem; 24 h, 10 μM); Reducing/scavenging agent: (N-acetylcysteine, Sigma; 10 h, 10 mM); Transcription inhibitors (Cordycepin, Sigma; 100 min, 50 μM; Alpha-amanitin, kindly provided by P. Janscak, 3–6 h, 20 μM); Ape1 inhibitor (Methoxyamine hydrochloride, Sigma; 10 h, 1 μM); CDK4/6 inhibitor (LY2835219, Selleckchem; 4 h, 1 μM); Cdc7 inhibitors (PHA-767491, Sigma; 8 h, 10 μM; XL-413, kindly provided by C. Santocanale, 4 h, 10 μM); CDK1 inhibitor (RO-3306, Sigma; 10 h, 10 μM); PLK inhibitor (BI-6727, Selleckchem; 4 h, 500 nM); Nucleosides (EmbryoMax, Millipore; 24 h, 5 ×). Roscovitine (Seliciclib, Selleckchem; 8 h, 20 μM).

350,000 cells were seeded in 6-well plates, 24 hours prior to drug treatment. Cells were treated with varying doses of ATR kinase inhibitor VE-821 (Selleckchem) or CHK1 inhibitor LY2603618 (Selleckchem) for 3 days at the end of which cells were counted to determine fractional cell survival. For dox-inducible EWS-FLI1 knockdown in A673 cells, shRNA was induced by treatment with 1μg/ml dox for 72 hours prior to ATR or CHK1 inhibitor treatment. For RNAseH1 experiments, A673 cells were infected with pLV-EF1a-RNAseH1-IRES-Blast lentivirus (derived from Addgene plasmid #85133) or empty vector and selected for stably infected cells.

For the dose–response relationship, chondrosarcoma cells were pre-treated with 2.5 µM, 10 µM, and 25 µM of the PARPi Olaparib or Veliparib, the ATMi Ku-55933, or the ATRi VE-821 (all Selleckchem, Houston, TX, USA). Afterwards, the cells were irradiated with 0 Gy (non-IR control), with a fractionated IR of 8 Gy X-ray on each of the three subsequent days, or with a total dose of 24 Gy. Cell viability was determined with the CellTiter-Glo^® cell viability assay (Promega Corporation, Madison, MI, USA) and normalized to the non-IR controls. Background reference values were derived from the culture media. Absorbance was measured with a LUMIstar™ microplate luminometer (BMG Labtech GmbH, Ortenberg, Germany) (mean ± SD; n = 3, performed in biological quadruplicates).
The xCELLigence RTCA-DP device (OLS, Bremen, Germany) was used to monitor cell proliferation in real-time. Cells were seeded after IR in electronic microtiter plates (E-Plate™, OLS) and measured for 120 h according to the manufacturer’s instructions. Cell density was measured in quadruplicate with a programmed signal detection every 20 min. Data acquisition and analyses were performed with RTCA software (version 1.2, OLS).

Kinase inhibitors of ATM (KU-60019, AZD1390, and AZD0156) and ATR (VE-821) were obtained from Selleck Chemicals (Houston, TX, USA). Sirolimus was obtained from LC Laboratories (Woburn, MA, USA). The concentrations and timepoints of the pharmacological inhibitors used in all experiments were based on previous studies [26 (link),27 (link),28 (link),29 (link),30 (link),31 (link),32 (link),33 (link),34 (link)].