Figure 3.
Expression of WT ATP13A3 restores the CHO-MG phenotype.A, stable cell lines were generated by lentiviral transduction to overexpress WT (A3-WT) or a catalytically dead mutant (A3-DN) of ATP13A3. Expression of the viral vectors was verified by immunoblotting using an ATP13A3 selective antibody, while the loading was monitored by a selective antibody of the house-keeping protein, GAPDH. B, cells were treated with 5-μM BODIPY–polyamines for 90 min at 37 °C with or without 90 min of 1-mM BV before treatment to inhibit polyamine uptake. Uptake was measured in terms of the mean fluorescence intensity (MFI) up to 1 × 104 events (debris free) per condition on the flow cytometer (n = 5–9). C, cells were treated for 24 h with different doses of MGBG. Cell viability was assessed using CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS), and dose–response curves were plotted (n = 3). Data represent the mean ± SD (B) and individual data points (representing replicates) are overlaid on bar graph plots, or the mean ± SEM (C) (∗∗∗∗/€€€€p < 0.0001, ns = not significant, ∗∗∗∗versus CHO-WT + A3-WT, €€€€versus CHO-MG and CHO-MG + A3-DN). 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; MGBG, methylglyoxal bis-(guanyl hydrazone); PUT, putrescine.