FIG 2.

ATRA inhibits SARS-CoV-2 infection in human Caco-2 cells and human respiratory organoids. (A to C) ATRA restricted SARS-CoV-2 infection in human Caco-2 cells. Human Caco-2 cells were incubated with serial concentrations of ATRA and then infected with SARS-CoV-2 at an MOI of 0.01. The viral load was determined by qRT-PCR at 48 h after infection (A). The spike and nucleocapsid proteins in the infected cells were detected by Western blotting (B) and immunofluorescence staining (C). (B) The black arrowhead indicates the full-length spike protein; the white arrowhead indicates the cleaved form. (C) The nucleocapsid was stained with Alexa Fluor 546-conjugated anti-rabbit IgG. The nuclei were stained with DAPI. The stained cells were examined using a Zeiss LSM 880 Meta confocal microscope in multitrack mode. Scale bars, 50 μm. (D to I) Antiviral activity of ATRA in human respiratory organoids. (D and E) Characterization of the human alveolar and bronchiolar organoids. (D) Human alveolar organoids. The differentiated bronchiolar epithelium was indicated by staining with hematoxylin and eosin or specific antibodies labeling type I (PDPN staining) and type II (SPC staining) alveolar epithelial cells. (E) Human bronchiolar organoids. The differentiated alveolar epithelium was composed mainly of ciliated columnar cells (β-IV-tubulin staining), goblet cells (MUC5AC staining), and basal cells (p63 staining). Scale bars, 20 μm. (F to I) Incubation with ATRA reduced SARS-CoV-2 infection in human alveolar (F, G) and bronchiolar (H and I) organoids. Secreted viruses of infected alveolar (F) and bronchiolar (H) organoids were detected in the supernatant at 24 and 48 h postinfection. The viral load in alveolar (G) and bronchiolar (I) organoids was detected at 48 h postinfection. Each dot represents an organoid. The data are presented as the mean ± SEM. The viral load was normalized to human GAPDH. The qRT-PCR primers are described in Table S1. (A and F to I) The data were analyzed using an unpaired t test. (A to C and F to I) Experiments were performed independently with at least three biological replicates with comparable results.