<i>N</i>⁴-Acetylcytidine enhances synthetic mRNA translation yield and fidelity

Ethics Human peripheral blood was obtained from de-identified healthy donors through the NIH Clinical Center, Department of Transfusion Medicine, Research Blood Donor Program, under a protocol approved by the NIH Institutional Review Board (IRB no. 99CC0168). All donors provided written informed consent before participation. Cell culture HeLa cells (American Type Culture Collection (ATCC), CCL-2) were cultured in DMEM (Thermo Fisher Scientific, 10313021) supplemented with 2 mM L -glutamine (Thermo Fisher Scientific, 25030164) and 10% bovine calf serum (BCS, HyClone, SH30073.03; DMEM-BCS). THP-1 cells (ATCC, TIB-202) were cultured in RPMI 1640 (Thermo Fisher Scientific, 21870092) supplemented with β-mercaptoethanol (55 mM, Sigma, M3148), 2 mM L -glutamine and 10% FBS (Seradigm, FBS, 97068-085, RPMI-primary). THP-1 cells were differentiated into M0 macrophages through the addition of phorbol 12-myristate 13-acetate (PMA, 162 nM, Sigma-Aldrich, P1585) with 1.5 × 10 6 cells per 6-well plate (for protein) or 0.375 × 10 6 cells per 12-well plate (for RNA and luminescence) for 16–24 h. THP-1-derived M0 cells were cultured in RPMI-primary medium for 24 h before transfection. HeLa and THP-1 cells were not authenticated. MEFs from a frozen cryovial were thawed and cultured in DMEM with 15% FBS and 1% l -glutamine. …