Pcr fluorescence probing

The trachea and lung of challenged hACE2‐K18 mice were collected and homogenized with gentleMACS M tubes (Miltenyi Biotec, 130‐093‐236) in a gentle‐MACS dissociator (Miltenyi Biotec, 130‐093‐235). RNAs were extracted using RNeasy Mini Kit (QIAGEN, 4104) according to the manufacturer's instruction, followed by the qRT‐PCR to determine the viral RNA copies of different tissues utilizing a one‐step SARS‐CoV‐2 RNA detection kit (PCR‐Fluorescence Probing) (DaAn Gene Co., DA0931). The SARS‐CoV‐2 nucleocapsid (N) gene was cloned into a pcDNA3.1 expression plasmid for standards to generate a standard curve. The indicated copies of N standards were tenfold serially diluted from 10⁹ to 10² and proceeded to qRT‐PCR utilizing the same one‐step SARS‐CoV‐2 RNA detection kit to obtain standard curves. The reactions were carried out on a BioRad CFX according to the manufacturer's instructions. The viral RNA copies of each tissue were calculated into copies per µg total RNA and presented as a log₁₀ scale.

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.

Lung and Trachea of challenged hACE2 mice were collected and homogenized with gentleMACS M tubes (Miltenyi Biotec, 130‐093‐236) in a gentle‐MACS dissociator (Miltenyi Biotec, 130‐093‐235). RNAs were extracted using RNeasy Mini Kit (QIAGEN, 74104) according to the manufacturer's instruction, followed by the qRT‐PCR to determine the viral RNA copies of different tissues utilizing one‐step SARS‐CoV‐2 RNA detection kit (PCR‐Fluorescence Probing) (Da An Gene Co., DA0931). To generate a standard curve, the SARS‐CoV‐2 nucleocapsid (N) gene was cloned into a pcDNA3.1 expression plasmid for standards. The indicated copies of N standards were tenfold serially diluted from 10⁹ to 10³ and proceeded to qRT‐PCR utilizing the same one‐step SARS‐CoV‐2 RNA detection kit to obtain standard curves. The reactions were carried out on a QuantStudio 7 Flex System (Applied Biosystems) according to the manufacturer's instruction. The viral RNA copies of each tissue were calculated into copies per µg total RNA and presented as log10 scale.