Figure 4.

Depletion of ATP13A3 in CHO-WT cells causes a CHO-MG phenotype.A, stable cell lines were generated by lentiviral transduction to knock down ATP13A3 in CHO-WT cells. Efficiency of the knockdown was verified at the mRNA level with quantitative PCR (n = 3). 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 = 3). C, cells were treated for 24 h with different doses of MGBG. Cell viability was assessed using CellTiter 96 AQ_ueous One Solution Cell Proliferation Assay (MTS), and dose–response curves were plotted (n = 3). Data represent the mean ± SD (A and B) and individual data points (representing replicates) are overlaid on bar graph plots, or the mean ± SEM (C) (^∗p < 0.05, ^∗∗p < 0.01, ^{∗∗∗∗/€€€€}p < 0.0001, ^∗∗∗∗versus CHO-WT + A3-KD1, ^€€€€versus CHO-WT + A3-KD2). Analyses were performed using two-way ANOVA and Bonferroni post hoc corrections. A3, ATP13A3; A3-KD, knockdown of ATP13A3; BODIPY, boron dipyrromethene; BV, benzyl viologen; FKBPA1, FKBP prolyl isomerase 1A; MGBG, methylglyoxal bis-(guanyl hydrazone); miR-fluc, expression of microRNA against firefly luciferase; PUT, putrescine.