Fig. 1

Metabolic characterization and response to ouabain treatment on [Na⁺]_i. a Principal component analysis of intracellular metabolites (mean values given in Supplementary Table S2). Intracellular concentrations of b lactate (left panel) and c phosphocholine (right panel) were significantly higher in all cancer cells with respect to control epithelial cells (n = 5). Extracellular metabolite concentrations of d glucose, e glutamine and f lactate after 24 h cell culture. Respective metabolite concentration from fresh media were subtracted such that negative concentrations refer to metabolite consumption while positive concentrations refer to production (n = 5). g MTT cytotoxicity assay dose response curves following 24 h treatment with ouabain. Measured EC₅₀ values were: 4T1: 40 µM, MDA-MB-231: 0.4 µM, HCC1954: 0.2 µM, MCF-7: 0.04 µM. h Cell viability in response to 1 µM ouabain for 1 h measured by trypan blue exclusion assay measured no change in cell viability. i Representative TQF ²³Na NMR spectra showing proportionality with cell number. The Tm-DOTP reference peak is from the internal standard. j Quantification of TQF ²³Na NMR relative to cell number and cell volume. Baseline [Na⁺]_i was higher in all cancer cells with respect to control epithelial cells (n = 5). Treatment with 1 µM ouabain for 1 h led to a significant increase in [Na⁺]_i in all human cancer cell lines compared to vehicle control (n = 5). [Na⁺]_i was unchanged in the murine 4T1 cell line following 1 h treatment with 1 µM ouabain, (p = 0.7, n = 5). Significance was assessed using a two-tailed unpaired t-test, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data plotted as mean ± SD