GoKawiilEthics
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 × 106 cells per 6-well plate (for protein) or 0.375 × 106 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. Primary monocytes were isolated from human peripheral blood using an EasySep Direct Human Monocyte Isolation kit (Stem Cell Technologies, 19669) according to the manufacturer’s instructions, followed by the addition of 25 mM HEPES (Quality Biological, 118-089-721), 50 ng ml–1 recombinant human IL-4 (Peprotech, 200-04) and 50 ng ml–1 recombinant human GM-CSF (Sigma-Aldrich, GF-304) in RPMI primary medium to generate MoDCs.
The SINAP technology relies on several auxiliary proteins: scFv–sfGFP to label the nascent peptides, MCP–RFP to label the RNA and the E3 ligase TIR1 from Oryza sativa (OsTIR1) for the auxin-inducible degron to deplete the mature proteins29. Two U-2 OS cell lines expressing these auxiliary proteins were used in this study: one for live-cell imaging (scFv–sfGFP, OsTIR1 (ref. 51) and MCP–RFP–CAAX), and the other for fixed-cell (scFv–sfGFP, OsTIR2 (ref. 52)) experiments. These U-2 OS cells (ATCC, HTB-96) were maintained in DMEM–FBS (Corning, 10-013-CM and Millipore Sigma, F4135), 100 U ml–1 penicillin and 100 µg ml–1 streptomycin (Millipore, P0781). Cells were cultured at 37 °C in a 5% CO 2 incubator and passaged approximately every 3 days. Monthly mycoplasma contamination testing was performed to ensure sterility.
Flow cytometry
The following antibodies were used for flow cytometry experiments: PE-conjugated anti-Hu/NHP CD25 antibody ((CD25-4E3), eBioscience, 12-0257-42), APC-conjugated anti-HuCD14 antibody ((61D3), eBioscience, 17-0149-42), PerCP/cyanine5.5-conjugated anti-human CD11c antibody ((3.9), BioLegend, 301624) and PE-conjugated anti-CD209 (DC-SIGN) antibody ((9E9A8), BioLegend, 330105). In brief, 0.1–1 × 106 cells were stained in 100 µl staining buffer (1% FBS in PBS) for 30 min at room temperature in the dark, washed twice with PBS and resuspended in 1% paraformaldehyde containing staining buffer. Flow cytometry acquisition was performed on a BD FACSymphony A5 and data were examined using FACSDiva software (v.9.3.1, BD Bioscience). Data were further analysed using FlowJo (v.10.8.1, BD).
Cloning
To generate a 5×C-stretch in the N-terminally Flag-tagged FLuc sequence3, a PCR strategy was used to introduce synonymous mutations of proline 197 and 198 (Supplementary Table 1). After confirmation of the mutation by Sanger sequencing, WT-5×C FLuc was back cloned into the original (WT-5×U FLuc) plasmid with BsrGI (NEB, R3575) and Bsu36I (NEB, R0524). Single-nucleotide insertion next to the C-rich sequence was accomplished by inserting a mutated DNA fragment (IDT) using BsrGI and Bsu36I. The sequence for the C=U NanoLuc reporter was purchased as a DNA fragment (IDT) and cloned using BbsI-HF (NEB, R3539) and BsmI (NEB, R0134).
In vitro transcription and polyadenylation
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