Azd1152 hqpa

Human primary hepatocytes and plating media were purchased from In Vitro Technologies (Baltimore, MD; product no. M00995-P; lot no. FOS). Recombinant human insulin and glucagon, fetal bovine serum (FBS), and sodium pyruvate were from ThermoFisher Scientific (Waltham, MA). Bovine serum albumin (BSA) was from EMD Millipore (Billerica, MA). Sodium lactate, glycerol, lysine, GW3965, SB277011-A, BMS753, AZD1152HQPA, A740003, and A841720 were from Sigma-Aldrich (St. Louis, MO). SB-742457, WAY100635, Ab142089, and MK7123 were from Nanjing Norris Pharm Technology Co., Ltd (Nanjing, China). SNX482, A 784168, CD3254, SB205607, AMTB, and ICI182,780 were from R&D Systems (Minneapolis, MN). SL0101 and SB269970 were from Merck Chemicals (Shanghai, China).

U2OS (ATCC, Manassas, VA, USA), U2OS-CASP2^−/−, U2OS-CASP2^−/−/CASP3^−/−, A549, A549-CASP2^−/− and stable cell lines were maintained in Dulbecco’s Modified Eagles Medium (DMEM, Sigma-Aldrich) supplemented with 10% foetal bovine serum (JRH Biosciences, Lenexa, KS, USA), 0.2 mM l-glutamine (Sigma-Aldrich), 15 mM HEPES (Sigma-Aldrich) and 100 µM penicillin/streptomycin (Sigma-Aldrich) in a humidified incubator at 37 °C with 10% CO₂. A549 and A549-CASP2^−/− cell lines were provided by Prof. Villunger (Medical University of Innsbruck, Austria). Where indicated, cells were treated with 100 nM BI2536 (Axon Medchem, Netherlands), 2 µM ZM447439 (Sigma-Aldrich), 400 nM AZD1152-HQPA (Sigma-Aldrich), or 50 µM blebbistatin (Selleck Chemicals, Houston, TX, USA) for 24 or 48 h. MEFs were maintained in DMEM (Sigma-Aldrich) supplemented with 0.2 mM l-glutamine (Sigma-Aldrich), 15 mM HEPES (Sigma-Aldrich), 10% foetal bovine serum (JRH Biosciences) and 50 µM β-mercaptoethanol (Sigma-Aldrich), non-essential amino acid mix (Sigma-Aldrich), 100 µM penicillin/ streptomycin (Sigma-Aldrich). Cells were cultured in a humidified incubator at 37 °C with 10% CO₂. Transfection of plasmid DNA was performed using Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. All cell lines were tested for mycoplasma contamination.

AZD1152-HQPA, the active metabolite of AZD1152, was obtained from Sigma-Aldrich (USA) and was dissolved in DMSO to incubate with the designated cells.