Extended Data Fig. 4: UDCA and ZGG require FXR to reduce ACE2 and SARS-CoV-2 infection.

(a–b) Immunofluorescence (a) and QPCR (b) showing downregulation of FXR expression following FXR knockdown (KD) in COs. Scale bars 25 μm. Housekeeping gene HMBS. One-way ANOVA adjusted for multiple comparisons; n = 4 independent experiments; centre line, median; box, interquartile range; whiskers, range; bars, standard deviation. (c) QPCR on FXR KD COs showing no change in the expression of ACE2 and the FXR downstream effector SHP following treatment with CDCA, UDCA or ZGG, demonstrating that FXR is indispensable for regulating ACE2 and SHP through these compounds. ACE2 and SHP expression in wild-type organoids shown in Fig. 1c and Extended Data Fig. 5a. Housekeeping gene, HMBS; n = 4; one-way ANOVA adjusted for multiple comparisons; ns, non-significant; centre line, median; box, interquartile range; whiskers, range; bars, standard deviation. (d) QPCR quantifying SARS-CoV-2 RNA 24 h post infection in FXR KD COs treated with CDCA, UDCA or ZGG. SARS-CoV-2 infection in the presence of FXR is shown in Fig. 1e. Housekeeping gene, GAPDH; n = 4 independent experiments; one-way ANOVA adjusted for multiple comparisons; centre line, median; box, interquartile range; whiskers, range; bars, standard deviation. (CDCA, UDCA and ZGG concentration, 10 μM). (e) Schematic illustrating the luciferase reporter construct containing the FXRE IR-1 identified in Fig. 1a and the mutagenesis strategy used in panel (f). (f) Luciferase reporter assay in HEK293 cells showing the transcriptional activity associated with the IR-1 located in the ACE2 promoter upon treatment with CDCA, UDCA or ZGG. Site-directed mutagenesis on IR-1 abolishes FXR binding/transactivation confirming the specificity of FXR binding on the ACE2 IR-1. IR-1 located in the SHP promoter used as positive control. N = 3 independent experiments; one-way ANOVA adjusted for multiple comparisons; bars, standard deviations. (CDCA, UDCA and ZGG concentration, 50 μM).