Fig. 4

Human colon organoids revealed the binding of STEAP3 to the viral receptor. (A) Human colon organoids were derived from tumor specimens of patients with colon cancer. The expression of specific markers of human intestinal epithelial cells, including enterocytes (E-cadherin [E-cad]), goblet cells (mucin [MUC-2]), enteroendocrine cells (synaptophysin [SYP]), and stem cells (leucine-rich repeat-containing G-protein-coupled receptor 5 [LGR5]), was detected and visualized in these organoids using immunofluorescence staining with the indicated antibodies, followed by confocal microscopy and three-dimensional image reconstruction. Scale bar = 100 μm. (B) The expression levels of STEAP3, the viral receptor ACE2, and specific markers of human intestinal epithelial cells in Caco-2 cells and human colon organoids were analyzed using immunoblotting with the indicated antibodies. (C) The total cell lysate was extracted from human colon organoids and subjected to IP assay with anti-ACE2 or anti-IgG antibodies. The binding of ACE2 to STEAP3 was further analyzed using anti-STEAP3 antibody. (D) Colocalization between ACE2 and STEAP3 in colon organoids was detected by double immunofluorescence staining with anti-ACE2 (green) and anti-STEAP3 (red) antibodies and visualized using an Andor Dragonfly confocal microscope. Scale bar = 25 μm. The highlighted area in the inset (arrow) was magnified two-fold. Scale bar = 20 μm. The uncropped and unprocessed immunoblot images are presented in Supplementary Fig. S18.