Fig. 4.

Innate immune response and SARS-CoV-2 (co)receptor expression in tonsil epithelial organoids. (A) Human cytokine array for secreted proteins from the tonsil organoids before (−) and after (+) LPS treatment at a concentration of 10 μg/mL for 24 h. (B) Changes in cytokine secretion levels were determined by densitometric analysis of the blots shown in (A). Values relative to the control levels (- LPS) are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by two-way ANOVA with Sidak's pairwise multiple comparison test. ***, p < 0.001; ****, p < 0.0001. n.d., not detected. A.U., arbitrary units. (C) Effect of LPS-stimulated culture supernatants on cell migration. HL-60 cells were treated with mock- or tonsil organoid culture medium before (- LPS) and after LPS stimulation (+LPS). Numbers of migrated cells are presented as means ± SEM of the three independent experiments. Two-way ANOVA with Sidak's multiple comparison test was used for statistical analysis. n.s., not significant. *, p < 0.05. (D) Immunofluorescence staining of ACE2 (left), TMPRSS2 (middle) and furin (right) (red) expressed in tonsil organoids on day 15 as well as in tonsil tissues. NGFR and MUC1 (green) are stained as epithelial markers for the basal and suprabasal/superficial layers, respectively. Merged images with Hoechst 33342 staining (blue) are presented on the right side of each panel. Zoom images of white rectangles are displayed at the bottom. Scale bars are 100 μm. (E) Quantitative RT-PCR to detect ACE2 (left), TMPRSS2 (middle) and Furin (right) mRNA transcripts on days 5, 10 and 15 in tonsil organoids originating from three different donors. Statistical analysis was performed by ordinary one-way ANOVA with Tukey's multiple comparison test. n.s., not significant. *, p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)