Cytation 3 microplate reader

The luciferase activity assays were carried out in three biological replicates (n = 3). The luciferase protein activity was assayed using the luciferase assay system kit (catalog no. E1500; Promega, USA) with modifications. Briefly, the yeast strains were grown for 8 h at 30°C in SC medium and harvested by centrifugation for 5 min at 4,000 × g. The cell pellet was disrupted using 200 µl of 2× lysis buffer reagent (Promega, USA) and 200 µl of glass beads in a TissueLyser II equipment (Qiagen, USA) for 3 min. Cells were centrifuged for 5 min at maximum speed, and the supernatant containing the protein extract was recovered and quantified by the Bradford standard method. The luciferase activity was assayed combining 5 µl of the total extracted proteins plus 100 µl of luciferase assay reagent (Promega, USA). The luminescence was immediately recorded in a Cytation 3 microplate reader (BioTek, USA), and it was normalized using the total protein concentration of each sample.

These assays were carried out as previously described^{13 (link)}. Briefly, cells were co-transfected with a 1:0.025:1:1 mixture of pGL4.37 E364A [(ARE:firefly luciferase) Promega]:pGL4.75, E693A [(CMV:Renilla luciferase) Promega]:pCS2 + 8 Halo-TEV-Keap1:pcDNA3 myc-Nrf2 with Mirus 2020 following the manufacturer’s protocol for 24 h. Cells were then subjected to T-REX as described above and incubated for a further 18 h. Media were removed and cells were lysed for 15 min in passive lysis buffer (Promega) with gentle shaking and homogenized lysate was transferred to the wells of an opaque white 96-well plate (Corning). Firefly and Renilla luciferase activity were measured sequentially on a BioTek Cytation3 microplate reader.

This assay was performed as described previously [10 (link)] but with some modifications. Fifty microliters of each sample were mixed with 200 µL of 450 µmol/L DPPH solution, and then, the mixture was incubated at 30 °C for 20 min. Subsequently, each mixture was centrifuged at 1500 rpm for 5 min at 15 °C. Then, 150 µL of each supernatant was placed per well in a 96-well plate (Costar^® 3595, Corning Incorporated, New York, NY, USA). The absorbance response was monitored at 517 nm for each sample in wells using the Cytation™ 3 microplate reader and Gen5™ software version 2.06 (Biotek Instruments Inc., Winooski, VT, USA). The results of this assay were expressed as the percentage inhibition of the radical or µg of ascorbic acid equivalents (AAE)/mL of sample. The percentage of inhibition against the DPPH radical was calculated using the absorbance values of the blank (distilled water) and experimental samples. The value expressed in µg AAE/mL of samples was obtained by interpolating the response of diluted samples into ascorbic acid calibration curves ranging from 6.0 to 120.0 µg/mL. For these experiments, ascorbic acid and distilled water were included as the positive and negative controls.

To detect the formation of syncytia, HEK293T cells expressing split GFP1-10 or GFP11 (Kindly provided by Prof. Dr. Oliver Schwarz, the Pasteur Institute, France^{62 (link)}) were mixed 1:1 at the final density of 6 × 10⁵ cells/mL. Then 500 µl cells were co-transfected with 350 ng of ACE2 and 350 ng of Spike expressing vectors using LT1. Forty hours post-transfection, fluorescence microscopy images were acquired using the Cytation 3 microplate reader (BioTek Instruments), and the GFP area was quantified using Fiji ImageJ.

For Supplementary Fig. 11, bacteria cells containing different CadC-TcpP promoter variants (with P5-TcpH) were streaked on LB plus 1.5% agar plates and grown overnight at 37 °C. Threes different colonies were inoculated into 1 mL of LB plus antibiotics in a 2 mL 96 deep-well plates (Thermo Fisher Scientific, 278606) sealed with AeraSeal film (Sigma-Aldrich, A9224-50EA) and incubated at 37 °C for 16 h with shaking (300 r.p.m.) and 80% of humidity in a Kuhner LT-X (Lab-Therm) incubator. After overnight growth the cells were diluted 1:250 into 200 µL 96-well plates with M9 minimal medium with 0.4% glycerol plus different TCA concentration. Measurements were done on a Cytation3 microplate reader (Biotek Instruments, Inc.). Absorbance at 600 nm and GFP (excitation 485 nm, emission 528 nm, gain 80) were measured every 10 min for 4 h (linear range of growth). Data were collected using a Gen5 Microplate Reader and Imager Software version 3.03. Raw data were processed by subtracting autofluorescence and normalizing by OD values. Michaelis–Menten enzyme kinetics was evaluated by using GraphPad Prism (version 8.0.2) nonlinear regression model.

HEK293 T cells were seeded in six-well format and transfected with 3 µg pBelo-SARSCoV-2-dSpike-GLuc-K2 or pBelo-SARSCoV-2-dSpike-EGFP and 0.25 µg of each expression construct pLVX-EF1alpha-SARS-CoV2-N-2xStrep-IRES-Puro, pCG-ACE2, pCAG-T7-RNA-polymerase, and one pCG- vector encoding the spike protein of SARS-CoV-2, RaTG13 or the indicated mutant S respectively. Two days after transfection, bright field and fluorescence microscopy (GFP) images were acquired using the Cytation 3 microplate reader (BioTek). Gaussia luciferase activity in the supernatants was measured with the Gaussia Luciferase Assay system (Promega) according to the company’s instructions.