Cordycepin

U20S were retrieved from ATCC and modified with a plasmid encoding for the restriction enzyme (pBABE-AsiSIER and pAID-AsiSIER)^{29 (link),30 (link)}. U2OS, DIvA (AsiSI-ER-U20S), and AID-DIvA (AID-AsiSI-ER-U20S) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogen) and either 1 µg/mL puromycin (DIvA cells) or 800 µg/mL G418 (AID-DIvA cells) at 37 °C under a humidified atmosphere with 5% CO₂. The cell lines were regularly checked for mycoplasma contamination. For AsiSI-dependent DSB induction, cells were treated with 300 nM 4OHT (Sigma, H7904) for 4 h. When indicated, 4OHT-treated cells were washed three times in pre-warmed PBS and further incubated with 500 µg/mL auxin (IAA) (Sigma; I5148) for the indicated time. For transcriptional inhibition, DRB (Sigma, 100 μM) or cordycepin (Sigma, 50 µM) was added to the medium 1 h prior to 4OHT (4 h) and auxin (2 h) treatments. Cells were arrested in G1 using a 48 h treatment with 40 μM lovastatin (Mevinolin from LKT Laboratories) and in G2 with a 24 h treatment with 40 μM Ro-3306 (CDK1 inhibitor, Calbiochem). For clonogenic assays in U20S cells, DSBs were induced either by increasing doses of etoposide (Sigma) for 16 h as indicated or by irradiation with a Cs137 source (Biobeam 8000).

Cordycepin and cisplatin were purchased from Sigma (St. Louis, Missouri).

Cordycepin (3’-deoxyadenosine) was determined in GRC and GRC-SC11 using HPLC (Agilent 1100 liquid chromatography system: Palo Alto, CA, USA). The measurement data were provided by the Korea Basic Science Institute (KBSI) on the Ochang Campus, South Korea. Waters Acquity BEH C18 columns (100 mm × 2.1 mm id, 1.7 µm and 12.5 mm × 4.6 mm id, 5 µm) were used. Water and methanol were used as mobile phases with a flow rate of 1.0 mL/min. UV detection was performed at the 254 nm wavelength. A calibration curve was prepared by plotting the peak area against the concentration using different concentrations of Cordycepin (Sigma-Aldrich, St. Louis, MO, USA).

Cordycepin was purchased from Sigma-Aldrich (Saint Louis, MO). The Lu1205, A375, GFP-tagged Lu1205, GFP-tagged A375, RFP-tagged Lu1205 and RFP-tagged A375 melanoma cell lines (obtained from ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% FBS and 100 U/ml of penicillin-streptomycin. All cells were maintained in a humidified incubator at 37°C and 5% CO₂.

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).

Adenosine and cordycepin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The hematoxylin and eosin (HE) stain kit (CATA: ab245880) and periodic acid Schiff (PAS) stain kit (CATA: ab150680) were purchased from Abcam (Cambridge, MA, USA).

N. benthamiana leaves were agroinfiltrated as described for the NMD suppression assay. After 3 days, leaves were infiltrated with buffer containing 1 mM PIPES (pH 6.25), 1 mM sodium citrate, 1 mM KCl, 15 mM sucrose and 0.08% Silwet L-77 with or without cordycepin (150 mg/ml, Sigma)^{70 (link)}. Immediately afterwards, leaves were detached and soaked in the equivalent buffer with or without cordycepin. For RNA and protein analysis, samples were collected at the time points 0 and 1 hour of incubation.