Figure 3: SLC39A14 isoforms 1 and 2 show differences in tissue expression, Mn uptake and transcriptional regulation.
From: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism–dystonia
(a) Immunostaining for SLC39A14 (all isoforms) in healthy control liver shows cell membrane expression (yellow arrow) and punctate cytoplasmic staining (black arrow); scale bar, 50 μm; Abcam anti-SLC39A14 antibody (ab106568, 1:100); and in globus pallidus (GP) from a healthy control shows positively stained large neurons (black arrow); scale bar, 100 μm; Novus anti-SLC39A14 antibody (NBP1-81551, 1:1,000). (b,c) Confocal images demonstrating the subcellular localization of fluorescently tagged human SLC39A14 isoform 1 (b) and isoform 2 (c) expressed in zebrafish embryos. Immunostaining for EGFP and mCherry at 6 hpf shows that both isoforms are expressed at the cell membrane (co-localization with membrane mCherry) and in the cytoplasm. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear stain. Scale bar, 50 μm. (d) RT–PCR of adult and fetal human tissues showing differences in mRNA expression between isoform 1 (ubiquitous expression in the tissues examined) and isoform 2 (* absent expression in brain, heart, skeletal muscle and skin). Amplicons for isoform 1 and 2 span 139 bp and 109 bp, respectively. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as a housekeeping gene. L, 100 bp ladder (Promega). (e) Graph showing Mn uptake in HEK-293 cells transiently transfected with wild-type SLC39A14 isoform 1 and 2, and empty pCS2+ vector following 15 and 30 min of MnCl2 (1 μM) exposure. Both isoforms facilitate Mn uptake. Cells transfected with isoform 2 have significantly higher Mn levels (P=0.009). Data are presented as means±s.d. from two independent experiments. Statistical analysis was performed using one-way ANOVA (P=0.0002 (15 min), P=0.0002 (30 min)) and Tukey’s multiple comparison test (***P<0.001). (f) Graph showing slc39a14 transcript levels assessed by qRT–PCR in 5 dpf zebrafish larvae after exposure to 500 μM MnCl2 for 24 h. Overall transcript levels are increased (P=0.035). Transcript levels of isoform 1 are unchanged (P=0.41) while those of isoform 2 show a 4.6-fold increase (P=0.005). Data are presented as means±s.d. from three independent experiments. Statistical analysis was performed using Student’s two-tailed t-test on individual ΔCt values (*P<0.05, **P<0.01). ANOVA, analysis of variance.
