Dulbecco s modified eagle s medium

A human Cervical Carcinoma Cell line (HeLa, ATCC CCL-2) and human Osteosarcoma epithelial cell line (U-2 0S, ATCC HTB-96) were grown in DMEM (Dulbecco’s modified Eagle‘s medium; Dominique Dutscher, Brumath, France) that contained 10% fetal bovine serum (FBS, Dominique Dutscher), at 37°C in a 5% CO2 atmosphere and 95% humidity. Cells were transiently transfected using JetPEI (Polyplus transfection kit, Ozyme, France). Lipofectamine RNAimax reagent (Fischer Scientific) was used to transfect siRNAs. cIAP1 si-RNA oligonucleotides were designed and purchased from Qiagen S.A.S. (Courtaboeuf, France). Cells were treated with 17nM GDC-0152 or 1μM Birinapant (Selleckmed, Euromedex, Souffelweyersheim, France) for 1hour, with cycloheximide (CHX) 100μg/mL (Sigma-Aldrich, St Quentin Fallavier, France) for up to 16 hours. MG132 (Sigma-Aldrich) was used at 1μM or 5μM overnight. Cells were synchronized using a 2 mM thymidine (Sigma-Aldrich) double block.

Human cervical carcinoma cell line (HeLa), human osteosarcoma epithelial cell line (U20S) and mouse embryonic immortalized (SV40) fibroblasts (MEF) cIAP1^−/−/cIAP2^−/− (J Silke, Melbourne, Australia), were grown in Dulbecco’s modified Eagle‘s medium (Dominique Dutscher, Brumath, France) or RPMI 1640 (Roswell Park Memorial Institute, Dominique Dutscher) containing 10% fetal bovine serum (Dominique Dutscher) at 37 °C in a 5% CO₂ atmosphere and 95% humidity. Cells were treated with 10 or 20 μM etoposide (Sigma-Aldrich, Lyon, France) for 6 h, 100 μg/ml cycloheximide (Sigma-Aldrich), 10–35 μM PYR-41 (Calbiochem, Merck Millipore, Saint-Quentin en Yvelines, France) for 24 h, 0.1–100 μM AMI-I (Enzo Life Sciences, Villeurbanne, France) for 24 h, or 1 μM GDC-0152 (Selleckmed, Euromedex, Souffelweyersheim, France). MG132 (Sigma-Aldrich,) was used at 1 μM or 5 μM overnight.

MDA-MB-231 (human mammary adenocarcinoma), A549 (human lung adenocarcinoma), SK-HEP-1 (human hepatocellular carcinoma), DU145 (human prostate adenocarcinoma) and the two human colon adenocarcinomas HCT116 and SW480 cell lines were cultured with high-glucose Dulbecco's modified Eagle's medium provided by Dutscher (Dutscher, Brumath, France) supplemented with 10% fetal bovine serum (Dutscher). PANC-1 (human pancreatic carcinoma) cell line was cultured in RPMI 1640 (Roswell Park Memorial Institute medium) provided by Dutscher and supplemented with 10% fetal bovine serum. All these cell lines were grown in an incubator at 37 °C and 5% CO₂.