Fig 1: Loss of slc39a14 function in zebrafish leads to increased Mn accumulation and sensitivity as well as impaired locomotor behaviour.(a) Mn levels assessed in homozygous slc39a14U801 and wild-type (WT) larvae show that mutant larvae have significantly raised Mn levels at 5 dpf (P=0.001) and 14 dpf (P=0.0002), and Mn accumulation on MnCl2 exposure (50 µM from 2 dpf) is significantly higher in mutant compared with WT larvae (P=0.000) at 5 dpf. Measurements were taken from pools of 10 larvae. Data are presented as means±s.d. from a minimum of five independent experiments. Statistical analysis was performed using Student's two-tailed t-test (***P<0.001). (b) Graph showing Fe, Zn and Cd levels in 14 dpf mutant and WT larvae. Levels of all three metals are not significantly different between the two groups (P=0.906 [Fe], P=0.257 [Zn], P=0.834 [Cd]). Measurements were taken from pools of 10 larvae. Data are presented as means±s.d. from five independent experiments. Statistical analysis was performed using Student's two-tailed t-test (NS, not significant). (c) Graph presenting the lethality in homozygous slc39a14U801 and WT larvae at 5 dpf on MnCl2 exposure between 2 and 5 dpf. Median lethal concentration (LC50) of MnCl2 determined by Probit regression analysis was 661 µM for WT (95% confidence interval (CI) 548–808 µM) and 377 µM (95% CI 313–455 µM) for mutant fish. Data are presented as means±s.e.m. from nine independent experiments. (d) Locomotor behaviour studies of homozygous slc39a14U801 and WT larvae show that in unexposed conditions there is no significant difference in locomotor activity; and on MnCl2 exposure, locomotor activity is markedly reduced in mutant larvae compared with WT. The locomotor behaviour was tracked during 4 and 7 dpf using automated analysis software. s/min, movement in seconds per minute. Data are presented as means±s.e.m. 12 larvae were analysed per condition. Statistical analysis was performed using two way ANOVA (i, P=0.18; ii, P=0.000) (***P<0.001; NS, not significant). ANOVA, analysis of variance.
Fig 2: SLC39A14B abolishes the antiproliferation and proapoptosis effects of SLC39A14 knockdown on RCC cells. (a) Analysis of SLC39A14 protein expression in RCC based on GEPIA database. (b) RT-qPCR analysis of the expression of SLC39A14B in RCC tissues and adjacent normal tissues (n = 67). (c) Northern blot analysis of SLC39A14B expression in RCC tissues and adjacent normal tissues (n = 3). (d) RT-qPCR analysis of SLC39A14A and SLC39A14B expression in A498 and 786-O cells transduced with sh-SLC39A14 alone or combined with SLC39A14A vector or SLC39A14B vector. (e) The proliferation of A498 and 786-O cells transduced with sh-SLC39A14 alone or combined with SLC39A14A vector or SLC39A14B vector detected by EdU. (f) Flow cytometry detection of the cell cycle of A498 and 786-O cells transduced with sh-SLC39A14 alone or combined with SLC39A14A vector or SLC39A14B vector. (g) Flow cytometric analysis of apoptosis of A498 and 786-O cells transduced with sh-SLC39A14 alone or combined with SLC39A14A vector or SLC39A14B vector. *p < 0.05. n.s means no statistical significance. The cell experiments were conducted three times independently.
Fig 3: SLC39A14 mutations lead to cerebral Mn deposition associated with characteristic MRI brain appearances.(a) Schematic of SLC39A14 showing its eight TMDs (pink and blue cylinders) interlinked by intracellular and extracellular loops35. TMD II, III, IV and VII (pink) are postulated by the transmembrane protein topology prediction tool MemSatSVM (see URLs) to form a pore. The histidine-rich (HXHXHX) and metalloprotease motif (EEXPHEXGD) are highlighted in orange. Mutated amino-acid residues are indicated by red circles. (b) Pedigrees and sequence chromatograms of family A-E. Affected individuals are indicated by black shading. Squares represent males, circles females and a double line a consanguineous union. Mutated bases are boxed in black. For each family, the top chromatogram shows the wild-type SLC39A14 sequence and the hromatogram below the homozygous SLC39A14 mutation identified in the affected individuals. Parental studies for families A, B, C and E demonstrate that both parents are heterozygous carriers of the identified mutation. (c) Representative MRI brain images of patients with SLC39A14 mutations showing characteristic radiological features: individual C-II-2 aged 3 years and E-II-2 aged 17 years. Generalized T1-hyperintensity of the cerebral white matter, globus pallidus (yellow arrows) and striatum (blue arrows), pituitary gland (white arrows), dorsal pons (pink arrows) and cerebellum (turquoise arrows) can be observed. Hypointensity of the globus pallidus is also evident on T2 and T2*-weighted imaging (white dashed arrows).
Fig 4: SLC39A14 deficiency causes hypermanganesemia and neurodegeneration that responds to chelation treatment with Na2CaEDTA.(a) Liver MRIs of a patient with SLC30A10 deficiency, individual E-II-2 with SLC39A14 mutations and a control subject. The extensive signal hyperintensity on T1-weighted imaging caused by hepatic Mn deposition in SLC30A10 deficiency is absent in individual E-II-2. There is only a subtle degree of T1-hyperintensity when compared with the control subject. Signal intensity of the liver (yellow arrow) was compared with that of the spleen (blue arrow). (b) Brain histology from post-mortem examination of subject D-II-1. Sections of globus pallidus and dentate nucleus stained with hematoxylin and eosin (H&E) show marked neuronal loss with only occasional remaining neurons (arrow) accompanied by reactive astrocytosis (shown within the ribbon of the dentate nucleus (between arrows)). Scale bar, 100 µm. Luxol fast blue/cresyl violet stain of a section of the cerebral white matter demonstrates patchy loss of myelin associated with coarse vacuoles (arrow). Scale bar 200 µm. (c) Graph showing whole-blood Mn levels and urinary Mn excretion over four courses of Na2CaEDTA treatment in individual E-II-2. Arrows indicate timing of Na2CaEDTA courses (day 1, 34, 52 and 84). Administration of Na2CaEDTA causes a significant increase in urinary Mn excretion (red) accompanied by a drop in whole-blood Mn levels (blue).
Fig 5: All treatments has no effect on the levels of iron transporter and regulatory proteins.The effects of the pharmacological interventions on the levels of iron transporter protein: (a) divalent metal transporter 1 (DMT1); (b) ZRT/IRT-like protein 14 (ZIP14); the levels of iron regulatory protein (c) hepcidin, and (d) ferroportin in iron-overloaded rats. The full-length blots are presented in Supplementary Figures 1, 2 and 3.
Supplier Page from Abcam for Anti-SLC39A14/ZIP-14 antibody