Glowmax

Calcium flux analysis was performed using Fluo-4 AM calcium flux assay (Invitrogen) according to the manufacturer protocol. Briefly, on the day of analysis cells were loaded for 60 mins with 5 μM Fluo-4 dye in calcium flux buffer. Fluorescence signal was recorded using florescence plate reader (Glowmax, Promega) before (background) and after stimulation with 10 μM ionomycin. The data are normalized to the background signal.

To determine ATP levels of yeast cells, intracellular metabolites were extracted using hot ethanol. Briefly, 1 × 10⁸ cells were harvested after 42 h culture time at 16,000 g for 2 min at room temperature (RT) and resuspended in 0.5 ml of boiling ethanol (75% ethanol, 10 mM (NH₄)₂SO₄) and incubated in a thermomixer at 1,000 (motor speed) rpm for 3 min at 90°C. Residual cell debris was removed by centrifugation at 16,000 g for 20 min at −5°C, and 10 µl of the supernatant was taken for the subsequent determination of ATP levels using the ATP Determination kit (Invitrogen, Cat. No. A22066). Luminescence was assessed with a microplate reader (GlowMax, PROMEGA, delay time 2 s, integration time 10 s, and detection range 350–650 nm). Data were normalized to the number of cells, as determined by CASY Cell Counter Technology (Schaerfe System, Roche). At least four different clones were measured per strain and construct, each with two technical replicates that were pooled before statistical analysis. This experiment was repeated at least three times independently.

The growth rate of CD26+ and CD26− cells, were evaluated by cell counting kit-8 (CCK8) (Microtech, Naples, Italy). This kit works based on the reduction of dehydrogenase and the formation of formazan. The amount of produced formazan represents the number of live cells. First, 1 × 10⁴ CMLT1 and HL60 were added to 96 wells plate and various concentrations of IL-VX (ranging from 10 nM to 1 µM) were added. After 48 h, 10 microliters/well of CCK8 dye were added and cells were incubated for 3 h at 37 °C. After this step, absorbance at 450 nm was measured using GlowMax (Promega, Madison, WI, USA) instrument. In this experiment, untreated CMLT1 and HL60 were used as the control to obtain the percentage of cell growth following IL-VX treatment. Meanwhile, culture media was used as the background. To obtain the percentage of cell growth following protocol was used.
$\% cell growth=\frac{OD Treated cells-OD background}{OD untreated cells-OD background} \times 100$

To determine the cell viability, the activity of mitochondrial dehydrogenases was measured with MTT assays. First, the electroporation of suspended cells was performed following the protocol described above. Subsequently, after a 24 h incubation, the culture medium from 96 well plate was removed, and 100 μL of 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) in PBS buffer was added. After 3 h of incubation at 37 °C, a 100 μL acidified isopropanol (0.04 M HCl in the absolute isopropanol) was added to dissolve formazan crystals. Finally, the multiplate reader (GlowMax, Promega, Walldorf, Germany) was used to measure the absorbance at 570 nm. The results were expressed as the percentage of viable cells relative to untreated (control) cells.

Vero cells were seeded (12 000 cells per well) in a white opaque 96-well plate. After overnight incubation the cells were infected with SARS-CoV-2-NLuc at 0.01 multiplicity of infection (MOI). At 1 hour post infection (hpi) the working stock of the virus was replaced by multiple concentrations (20, 10 and 0.1 μM) of compounds. Remdesivir and DMSO were used as positive and negative controls respectively. At 24 hpi, 50 μL of Nano luciferase substrate (Promega) was added to each well and after 10 min of incubation at room temperature luciferase signals were measured using a Promega Glow Max microplate reader. The relative luciferase signal was recorded and plotted against compound concentration using software Prism.

ARPE-19 cells plated in a 96-well plate were treated (1) with or without FA for 3 days; (2) with or without FA for 1 h before a 1-h exposure to 0.3–0.4 mM H₂O₂ with fresh medium replaced every day for 3 days; (3) without drugs for 1 h before a 1-h exposure to 0.3–0.4 mM H₂O₂ with fresh medium containing FA or none replaced every day for 3 days. At the end of the incubation period, cell viability was assessed by WST-8 solution (Cell Count Reagent SF; Nacalai Tesque). The cells were incubated with WST-8 solution for 1–2 h and scanned at 450 nm with a microplate reader (Glow Max; Promega, Tokyo, Japan).