Luciferase assay system

p53 transcriptional activity was assessed using Ba/F3 JAK2V617F cells stably expressing the pGreenfire1^TM p53 reporter construct (Systems Biosciences) using the Luciferase Assay System (Promega). Details are in the Supplementary Information. In brief, cells were harvested during log phase growth and 6x10⁵ cells (at 2x10⁵/ml) treated with 5μM Nutlin or 100nM CBL0137 for 6 hours prior to analysis of reporter activity using the Luciferase Assay System (Promega) as per manufacturer’s instructions. Luminescence was measured on a FLUOstar Omega plate reader (BMG Labtech) using a 10-second measurement read for luciferase activity.

SARS-CoV-2 and variant pseudotypes were generated by cotransfection of the S-glycoprotein-encoding plasmid and human immunodeficiency virus (HIV) backbone expressing firefly luciferase (pNL4-3.luc.RE) into HEK293T cells (ATCC, CRL3216) using Lipofectamine 2000 transfection reagent (Life Technologies, 11668-019) according to the manufacturer’s instructions^{85 (link)}. Pseudotyped virus stocks were collected 48 h after transfection, filtered and stored at −80 °C. Viral titers were measured based on luciferase activity determined by relative light units (Luciferase Assay Systems, Promega Biosciences). Plasma samples or antibodies were serially diluted threefold and then incubated with SARS-CoV-2 pseudovirus for 1 h at 37 °C. HeLa-hACE2 cells (Prof. Qiang Ding, Tsinghua University; HeLa, ATCC CCL-2) (1.3 × 10⁴ per well) were directly added to the antibody-virus mixture. After 48 h, the cells were washed with PBS and lysed with Luciferase Cell Culture Lysis 5× reagent (Promega, E1531), and the luciferase activity was measured using the Luciferase Assay System (Promega, E1501). Plasma or mAbs were tested in duplicate wells, and the assay was independently repeated at least twice for sets of plasma samples and individual mAbs. The neutralization ID₅₀ or IC₅₀ was calculated using nonlinear regression (log [inhibitor] vs. normalized response, variable slope) (GraphPad Prism v.8.0).

HEK293, Hep3B, or CFBE41o^- cells were seeded in 96-well cell culture plates at 1 × 10⁴ cells/well and grown in Dulbecco’s Modified Essential Medium (DMEM, Pan Biotech or Gibco) for Hep3B or Minimum Essential Medium (MEM, Pan Biotech) for CFBE41o^- cells supplemented with 10% fetal bovine serum (FBS, Pan Biotech) and 2 mM l-glutamine (Thermo Fisher Scientific). 24 h later, cells were co-transfected in triplicate with 25 ng PTC-FLuc or WT-FLuc plasmids and 100 ng of IVT sup-tRNA or mismatched tRNA using lipofectamine 3000 (Thermo Fisher Scientific). After 4–6 h, the medium was replaced and 24 h post-transfection cells were lysed with 1x passive lysis buffer (Promega). Luciferase activity was measured with luciferase assay system (Promega) on Spark microplate reader (Tecan).
In some experiments, cells were treated with 5 µM eRF1 inhibitor (SRI-41315, MedChemExpress) and after 18 h transfected with sup-tRNA as described above for 6 h (total duration of eRF1 treatment was 24 h). Thereafter, cells were lysed with 1x passive lysis buffer (Promega). Luciferase activity was measured with luciferase assay system (Promega) on Spark microplate reader (Tecan).

Luciferase reporter assay was performed as described previously [26 ]. Sequences of 101 bp flanking rs17268364 were synthesized and subcloned into pGL3-promoter vector (Promega, Madison, WI, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the instruction of the manufacturer (sequences are shown in Supplementary Table S3). Luciferase reporter assays were determined using the Promega Luciferase Assay System (Promega, Mannheim, Germany).

HEP3B hepatoma and HEK293T cells were plated in 24-well plates at 2.5×10⁴ cells/well, and transfected the next day using Fugene 6, following manufacturer protocols (Roche Molecular Biochemicals, Burgess Hill, UK). Two different concentrations of pGL3-promoter plasmid were transfected, and pCMV-LacZ was co-transfected in all cells (pCMV-LacZ was kindly provided by J. Braganca and S. Bhattacharya). Luciferase activity was measured using Promega Luciferase Assay System (Promega, Southampton, UK), and read on a Labsystems Luminoskan Ascent microplate luminometer (Thermo Fisher Scientific, Loughborough, UK). Activity of β-galactosidase was assayed by measuring cleavage of the substrate o-nitrophenyl-β-D-galactoside (ONPG) [37] (link). Two to three independent transfection experiments were run, each time in triplicate wells, and results are corrected for transfection efficiency by expressing data as the ratio of luciferase to β-galactosidase activity.