Figure 5.
ATP13A3-dependent transport exhibits a broad specificity toward PUT, SPD, and SPM.A–D, cells were treated with only 5-μM BODIPY–PUT, BODIPY–SPD, or BODIPY–SPM (n = 4) (A), or with 5-μM BODIPY–PUT combined with different concentrations (1 μM, 5 μM, 10 μM, or 50 μM) of MGBG (n = 4) (B), nonfluorescent PUT (n = 3–6) (C) or nonfluorescent SPD/SPM (n = 3) (D) for 90 min at 37 °C. Uptake was measured in terms of the mean fluorescence intensity (MFI) up to 1 × 104 events (debris free) per condition on the flow cytometer. E–G, cells were treated for 72 h with the indicated concentrations of PUT (E), SPD (F), and SPM (G) combined with 1-mM DFMO. CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) was used to assess cell viability, and dose–response curves were plotted (n = 3). Data represent the mean ± SD (A–D), and individual data points (representing replicates) are overlaid on bar graph plots, or the mean ± SEM (E-G) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns = not significant). Analyses were performed using two-way ANOVA and Bonferroni post hoc corrections. A3-DN, overexpression of ATP13A3 catalytically dead mutant D498N; A3-WT, overexpression of WT ATP13A3; BODIPY, boron dipyrromethene; BV, benzyl viologen; DFMO, difluoromethylornithine; MGBG, methylglyoxal bis-(guanyl hydrazone); PUT, putrescine; SPD, spermidine; SPM, spermine.