Lightcycler 480 2 software version 1

The virus isolate has been described before [7 (link),35 (link)]. The cells were seeded in 48 well plates (Vero, 15,000/well; Huh-7, 30,000/well; Calu-3, 100,000/well). Then the cells were incubated with AKS461 and, after approximately 15 min, infected with SARS-CoV-2 (MOI = 1). The medium was exchanged to remove inactive viruses, which influence genome copy determination. All infection experiments were performed in triplicate assays and repeated at least twice in independent experiments. After 72 h, 200 µL of the medium was collected, and viral genomes were purified with the High Pure Viral Nucleic Acid kit (Roche, Mannheim, Germany). SARS-CoV-2 RNA genomes were quantified with the dual-target SARS-CoV-2 RdRP RTqPCR assay kit, containing universal SARS-CoV-2 primers and with viral RNA multiplex master kit (Roche, Mannheim, Germany) with the LightCycler 480 II (Roche, Mannheim, Germany). The provided standard was used for genome copy-number quantification using the LightCycler 480 II Software Version 1.5 (Roche, Mannheim, Germany). The PCR reactions were performed in duplicates.

The virus isolate has been described before [14 (link),20 (link)]. The cells were seeded in 48-well plates (Vero, 15,000/well; Huh-7, 30,000/well; Calu-3, 100,000/well). The next day the cells were incubated with the compounds and, after 10 to 15 min, infected with SARS-CoV-2. After 24 h, the medium was exchanged to remove inactive viruses since they would influence the genome copy determination. All infection experiments were performed in triplicate assays and repeated at least twice in independent experiments. After 72 h, 200 µL of the medium was collected, and viral genomes were purified with the High Pure Viral Nucleic Acid kit (Roche, Mannheim, Germany). SARS-CoV-2 RNA genomes were quantified with the dual-target SARS-CoV-2 RdRP RTqPCR assay kit, containing universal SARS-CoV-2 primers and with viral RNA multiplex master kit (Roche, Mannheim, Germany) with the LightCycler 480 II (Roche, Mannheim, Germany). The provided standard was used for genome copy-number quantification using the LightCycler 480 II Software Version 1.5 (Roche, Mannheim, Germany). The PCR reactions were performed in triplicates. Quantifications were performed with the respective cycler software. EC₅₀ values were calculated using GraphPad Prism Version 6 (GraphPad Software; Boston, MA, USA).

Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. qRT-PCR was performed using LightCycler® 480 II System with LightCycler® 480 SYBR Green Mix and data were analyzed with LightCycler® 480 II Software, version 1.5, all provided by Roche. Reactions were performed in triplicate for both the target and the housekeeping gene ribosomal protein L38 (RPL38) used for normalization. Relative quantification of the target gene expression was calculated by the comparative threshold cycle (Ct) method.