Seliciclib

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).

Primary samples were obtained with informed consent in accordance with Mayo Clinic IRB-approved research protocols and handled according to Good Clinical Practice. Primary cells were Ficoll-gradient separated, and immediately cultured, or viably frozen for subsequent short-term culture. Cell lines were obtained from ATCC or DSMZ. All cell lines tested negative for mycoplasma before cryopreservation, and all cell lines were confirmed free of cross-contamination using PCR-based DNA fingerprinting. Cells were cultured in RPMI-1640 containing 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin (Invitrogen) at 37°C/5% CO2. Compounds were obtained as follows: Alvocidib (Tolero Pharmaceuticals), venetoclax/ABT-199 (ChemieTek), ABT-737, LDC067, palbociclib, seliciclib and Ro-3306 (SelleckChem), LY2857785 (MedChem Express), NU6102 and JQ1 (Cayman Chemical).