Fig. 2: Inverted ALI culture model supports SARS-CoV-2 infection.

Immunofluorescent images of sections of ALI cultures stained for SARS-CoV-2 entry factors (magenta) and nuclei (Hoescht, cyan). Bottom panels show entry factor staining in inverted grayscale and brightfield. Cilia marked with arrowheads. Scale bar = 20 µm. Cultures from 2 donors were stained with comparable results. a ACE2. b TMPRSS2. c Copies of nucleoprotein (N) RNA per square millimeter of culture area in mucus collected from inverted (pink) and conventional (green) ALI cultures immediately prior to or at several time points after infection. 1 HPI = post-infection rinse, as an approximation of input virus. Points represent the mean of technical duplicates (n = 3–6 ALI cultures across 3 donors per time point per condition). Error bars represent the mean of means ± standard deviation. Significance by two-sided Welch’s t-test. d Immunofluorescent images of a section of an ALI culture 72 h after SARS-CoV-2 infection stained for SARS-CoV-2 N (magenta), Spike (S, green), double-stranded RNA (dsRNA, blue), and nuclei (Hoescht, white). Results are typical from 3 independent experiments. Thresholding of viral antigens is altered in the right inset panels to highlight dim viral protein puncta among the cilia and mucus. Yellow arrowheads point to cilia. Rightmost panels show single channels in inverted grayscale. Skinny yellow rectangles surround the lines profiled in (e and f). Scale bar = 10 µm in large image and 5 µm in insets. e, f Line profile pixel intensities of cellular (e) and mucus (f) viral antigen (magenta N, green S) and dsRNA (blue) staining. Source data for all plots are provided as a Source Data file.