Fig. 3: 4OI and SEL reduce ACE2 and XPO1 levels.

A–C Reduction of ACE2, TMPRSS2, and XPO1 mRNA. RT-qPCR analysis of RNA from the experiment shown in Fig. 1A–D. D 4OI and SEL reduce ACE2 and TMPRSS2 protein levels. Uninfected Calu-3 cells were grown in medium containing 4OI (100 µM) or SEL (1 µM), and cellular ACE2 and TMPRSS2 levels were measured by immunoblot after 1–72 h. E 4OI reduces half-life of ACE2. Uninfected Calu-3 cells were grown in medium containing 4OI with or without cycloheximide (CHX, 50 µg/ml), and cellular ACE2 levels were measured by immunoblot after 1–12 h. Densitometry of the blots shown in (E); F, bar chart; G, numerical values; untreated cells = 100%. H NEDD4L knock-down attenuates the ACE2-destroying capacity of 4OI at the protein. Efficiency of NEDD4L mRNA knock-down is shown in Supplementary Figure S2F. I. MDM2 knock-down attenuates the ACE2-destroying capacity of 4OI at the protein level. Efficiency of MDM2 mRNA knock-down is shown in Supplementary Figure S2G. J–N Uninfected Calu-3 cells were grown in medium containing 4OI, SEL, proteasome inhibitor (CFZ, 10 nM), or lysosome inhibitors (BAFA1- 100 nM, CQ- 50 µM), and expression of ACE2 was determined by immunoblot. CFZ 6 h (J), BAFA1 24 h (K, L), and CQ 24 h (M, N). O 4OI and SEL reduce XPO1 protein levels. Uninfected Calu-3 cells were grown in medium containing 4OI or SEL, and XPO1 levels were measured by immunoblot after 1–72 h. P, Q NEDD4L and MDM2 knock-down attenuate ACE2 mRNA reduction by 4OI and SEL (RT-qPCR after 24 h). R 4OI and SEL attenuate STAT3 phosphorylation (immunoblot after 24 h). n = 3 (D, E, H, I, O: n = 2), means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.