SARS-CoV-2 Variant Inhibition by DCM in ALI Cells

The ALI-cultured cells on the apical chamber of the transwell inserts were infected with the SARS-CoV-2 variants [including Alpha, Delta and Omicron (BA.1, BQ.1 and XBB.1)] viral inoculum at an MOI of 0.1. The inoculated plates were incubated for 1 h at 37 °C with 5% CO₂. At the end of the incubation, the inoculum was removed from the apical chamber. The basolateral side of the AECs culture was pre-treated with DCM at concentrations of 0–250 μM (dissolved in 0–1 mM NaOH) for 8 h prior to viral infection, and DCM was maintained in the media until 24 h post-infection.
The viral RNA was extracted from the cell supernatant (derived from apical of ALI culture) by using the EZ-press Viral RNA Purification Kit (EZB-VRN1, EZBioscience, Roseville, MN). SARS-CoV-2 N gene copy number was determined using a novel coronavirus (2019-nCoV) nucleic acid diagnostic kit (PCR-Fluorescence Probing) (Daan Gene, Guangzhou, China) according to the manufacturer’s protocol. The percentage of inhibition of SARS-CoV-2 N gene copies in supernatant of cultured AECs was estimated for each drug concentration, and the IC50 was determined by using Graphpad Prism (version 8.0).^{46 (link),47 (link)}In addition, the infected cells were harvested to assess N protein expression using SARS-CoV-2 antibodies via IF staining.