Figure 2.

Cardiac glycosides ouabain and digoxin inhibit kynurenine production and IDO1 protein expression in MDA-MB-231 and A549 cells.A, dose-response curve for ouabain and digoxin treatment of TNF/IFN-ɣ stimulated MDA-MB-231 cells. IC₅₀ ouabain = 89 nM, 95% confidence interval 70 to 112 nM. Apparent IC₅₀ digoxin ∼164 nM, n = 3. B, dose–response curve for ouabain and digoxin treatment of TNF/IFN-ɣ stimulated A549 cells. IC₅₀ ouabain = 17 nM, 95% confidence interval 14 to 20 nM. IC₅₀ digoxin 40 nM, 95% confidence interval 35 to 46 nM, n = 3. C, representative SBFI data from three independent experiments measuring intracellular sodium in response to 24-h cardiac glycoside or 0.05% DMSO control treatment of TNF/IFN-ɣ-stimulated MDA-MB-231 cells. ∗∗∗p < 0.001, ∗∗p < 0.01, One-way ANOVA, Tukey’s multiple comparisons. D, ouabain titration inhibits kynurenine production (3 independent experiments, •) and increases intracellular sodium ([Na⁺]_i (2 independent experiments, ▪) in TNF/IFN-ɣ stimulated MDA-MB-231 cells treated with the indicated concentrations of ouabain. Data were fitted using nonlinear regression by least squares fit. Pearson Correlation r² = 0.9628, p < 0.05. E, representative Western blots of IDO1 protein expression in TNF/IFN-ɣ-stimulated MDA-MB-231 and A549 cells pre-treated with ouabain or 0.05% DMSO. F, IDO1 and GAPDH protein expression in MDA-MB-231 (top) or A549 (bottom) cells pretreated with ouabain, digoxin, or 0.05% DMSO control for 36 h, then TNF/IFN-ɣ stimulated for 24 h. Ouabain and digoxin treated samples were visualized on two different blots. G, relative IDO1 band intensities determined from Western blots of three independent experiments in which TNF/IFN-ɣ-stimulated MDA-MB-231 cells were pretreated with 0.05% DMSO or 100 nM ouabain. H, IDO1 mRNA levels from three independent experiments in which TNF/IFN-ɣ stimulated MDA-MB-231 cells were pretreated with 0.05% DMSO or 100 nM ouabain. IDO1, indoleamine-pyrrole 2′,3′-dioxygenase 1; SBFI, sodium-binding benzofuran isophthalate.