Journal: Cardiovascular Research

Article Title: ATP13A3 variants promote pulmonary arterial hypertension by disrupting polyamine transport

doi: 10.1093/cvr/cvae068

Figure Lengend Snippet: ATP13A3 deficiency impairs polyamine transport in ECs. ( A ) Cellular PUT, SPD, and SPM levels in hPAECs measured by LC–MS. Cells were transfected with DharmaFECT 1™ (DH1, Cambridge, UK) alone, si ATP13A3 , or non-targeting siRNA control (siCP) and cultured overnight in EBM2 containing 2% FBS supplemented with or without 1 mM PUT, 10 µM, SPD, or 10 µM SPM. The data ( n = 3 experiments) are presented as polyamine peak area ratio relative to 2% FBS DH1. ( B ) Western blot showing ATP13A3 protein expression in parental, non-transduced, and HMEC-1 cells stably expressing miRNAs targeting ATP13A3 (miR1–miR4), with miR-FLUC (Firefly Luciferase) as a control. ( C ) BDP-labelled polyamine uptake in HMEC-1 stable knockdown lines ( n = 4 experiments, two technical replicates per experiment). The data are normalized to the mean fluorescent intensities of miR-FLUC. ( D ) Confocal microscopy depicting the uptake and distribution of PUT-BDP in HMEC-1 cells, expressing miR-FLUC and ATP13A3 miR3 following 2 h treatment with PUT-BDP (scale bar = 10 µm). ( A , C ) The data (mean ± SEM) were analysed using a one-way ANOVA with Tukey’s post hoc test for multiple comparisons. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: BODIPY-tagged spermine (SPM-BDP), spermidine (SPD-BDP), and putrescine (PUT-BDP) were synthesized, as previously described, and dissolved in 0.1 M 3-morpholinopropane-1-sulfonic acid (MOPS), pH = 7.0 (AppliChem, A1076, Darmstadt, Germany).

Techniques: Liquid Chromatography with Mass Spectroscopy, Transfection, Cell Culture, Western Blot, Expressing, Stable Transfection, Luciferase, Confocal Microscopy

Journal: Cardiovascular Research

Article Title: ATP13A3 variants promote pulmonary arterial hypertension by disrupting polyamine transport

doi: 10.1093/cvr/cvae068

Figure Lengend Snippet: The ATP13A3 LK726X frameshift variant predisposes BOECs to apoptosis by affecting ATP13A3-mediated polyamine transport. ( A ) Immunoblotting of ATP13A3 in control BOECs (C4, C7, C35) and ATP13A3 LK726X BOECs. Densitometric analysis of ATP13A3 and α-tubulin was performed (graph, n = 4 experiments). ( B ) Cellular polyamine contents, measured by LC–MS, of BOECs in media or supplemented with 1 mM PUT, 10 µM SPD, or 10 µM SPM. The data ( n = 3 experiments) are presented as polyamine peak area ratio relative to the C4 control BOEC line. ( C ) BOEC uptake of PUT-BDP, SPD-BDP, and SPM-BDP measured by flow cytometry ( n = 3 experiments, two technical replicates per experiment). ( D ) Cell apoptosis of BOECs cultured in EBM2 supplemented with 1% FBS or 5% FBS was assessed by Caspase-Glo®3/7 assay ( n = 5 experiments). The data are normalized to cells cultured in EGM2 containing 10% FBS. ( A–D ) The data are mean ± SEM analysed using ( A , B , D ) one-way ANOVA with Tukey’s post hoc test for multiple comparisons or ( C ) unpaired t -test. * P < 0.05, ** P < 0.01, **** P < 0.0001 compared with ATP13A3 LK726X .

Article Snippet: BODIPY-tagged spermine (SPM-BDP), spermidine (SPD-BDP), and putrescine (PUT-BDP) were synthesized, as previously described, and dissolved in 0.1 M 3-morpholinopropane-1-sulfonic acid (MOPS), pH = 7.0 (AppliChem, A1076, Darmstadt, Germany).

Techniques: Variant Assay, Western Blot, Liquid Chromatography with Mass Spectroscopy, Flow Cytometry, Cell Culture, Caspase-Glo Assay