Cell Culture and Transfection Techniques

HeLa cells, HEK293T cells and U2OS cells were maintained in DMEM (Life Technologies) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (Hyclone). Cells were transfected with 1 µg of total DNA per 4 × 10⁵ cells, unless indicated otherwise, using JetPrime (PolyPlus transfections) according to the manufacturer's instructions. If more than one plasmid was used to transfect cells, the amount of each plasmid used per transfection reaction was constant. Twenty-four hours after transfection, cells were fixed or lysed. For siRNA transfection, 20 nM of siRNA was used to transfect 150,000 cells using Lipofectamine 2000 (Invitrogen) reagent according to manufacturer's instructions. Cells were treated with 500 mM arsenite (Sigma-Aldrich) for 1 h and with 1 µM Emetine (Sigma-Aldrich) for 50 min (Cinti et al. 2017 (link)).