Figure 4.

Cardiac glycosides block the inhibitory effect of SARS-CoV-2 [S1S2] spike on cAMP-activated CFTR chloride channels in differentiated BCi.NS1.1 (d-BCi) epithelia and 16HBE14o-cells. (a) ALI differentiated dBCi cells were incubated apically with 400 ng/mL Wuhan-Hu-1 [S1S2] spike protein for 4 h in the presence of 30 nM ouabain, digitoxin or digoxin, washed and then incubated for additional 20 h under ALI conditions. CFTR-dependent short-circuit currents (I_sc) were measured in Ussing Chambers as the changes in response to Amiloride, IBMX/ Forskolin, and CFTR_inh-172. (a) Representative current I_sc tracings and (b) summary of changes in I_sc of four independent Ussing chamber experiments are shown along with a representative image of CFTR Western blot image of treated cells after Ussing chamber analyses (lower panel). (c) Submerged 16HBE14o- cell cultures were treated with 400 ng/ml β-1.351 [S1S2] spike protein in the presence or absence of 50 nm digitoxin, digoxin or ouabain for 4 h in Serum-free αMEM medium, then washed out with the serum-free αMEM medium, incubated in the complete αMEM medium for additional 20 h. CFTR quantitative analyses normalized with β-Actin values (top panel) and a representative CFTR Western blot image (lower panel) are shown. β-actin was used for equal loading of protein. The data are expressed as means ± SD (N = 3–4). Statistical p values were determined with a one-way ANOVA, followed by both Holm’s and Dunnett’s post-hoc tests comparing each mean to the medium control. For the Holm test, *, p < 0.05; and ¥, p < 0.01. Dunnett’s tests were consistently significant (p < 0.05), except as noted (•).