Ly2835219

Fascaplysin, PD0332991, and LY2835219 were purchased from Selleck Chemicals (Houston, TX, USA). Z-VAD-FMK, MG132, MAZ51, and cycloheximide were purchased from Sigma Aldrich (St. Louis, MO, USA). Recombinant TRAIL was purchased from PeproTech (Rocky Hill, NJ, USA). For Western blotting, antibodies recognizing phospho-mTOR (CST-5536), mTOR (CST-2983), phospho-4EBP1T70(sc-18092), phospho-4EBP1S65(sc-18091), phospho-4EBP1T37/T46(CST-2855), 4EBP1 (CST-9452), phospho-p70S6K1 (CST-9234), phospho-RB (CST-8516), cleaved-caspase-9 (CST-9505), cleaved-caspase-3 (CST-9664), cleaved-PARP (CST-5625), c-myc (sc-40), cyclin D1 (sc-8396), and β-tubulin (sc-9104) were purchased from Cell Signaling Technology (Danvers, MA, USA) and Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies recognizing survivin (AF886), and HIF-1α (ab1) were purchased from R&D systems, Inc. (Minneapolis, MN, USA) and Abcam (Cambridge, MA, USA).

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).

CDK4/6 inhibitors PD0332991 (S1116) and LY2835219 (S7158) were obtained from Selleckchem (Houston, TX) and stock solutions prepared in dimethylsulfoxide. They were added to cells 24 h prior to Western blotting or respiration analyses. For CDK4 knockdown, cells were transfected with siRNA targeting human CDK4 (Ambion #4390824, s2824) or a non-silencing control (Ambion #4390843) and Lipofectamine RNAiMax Transfection Reagent (Invitrogen #13778150) obtained from ThermoFisher Scientific (Waltham, MA) according to the manufacturer's protocol. Briefly, the cells were plated at about 60-80% confluency in a six well plate. The next day, the media was changed to Opti-MEM medium (ThermoFisher Scientific). The respective siRNAs (60pmole) were diluted to 150 μL in Opti-Mem and then separately mixed 1:1 with Transfection Reagent (9 μL diluted to 150 μL in Opti-Mem) and then incubated for 5 min at room temperature. The siRNA-lipid complexes were then added to the cells and after 6-8 hours of incubation, equal volumes of DMEM containing 10% FCS were added and cells harvested 72 h later.