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. 2014 Oct 15;34(42):14079–14095. doi: 10.1523/JNEUROSCI.2329-14.2014

Figure 4.

Figure 4.

Expression of SLC30A10–WT increases Mn efflux. A, HeLa cells were transfected with a control construct (Rab5; denoted as Control) or SLC30A10–WT. Two days after transfection, cells were treated with the indicated amounts of Mn for 16 h. The absolute level of intracellular Mn was then measured using ICP-MS and normalized to total protein. Data shown are from one representative experiment; error bars depict SDs from three ICP-MS runs. Analysis from three independent experiments is presented in B. B, Quantification of the difference in intracellular Mn between cells expressing control (Rab5; Control) or SLC30A10–WT constructs from multiple experiments. HeLa cells were transfected as described in A and, 2 d after transfection, treated with 500 μm Mn for 16 h. Intracellular Mn normalized to total cellular protein was then measured as described in A. In each experiment, for cells expressing the control construct, the absolute value of intracellular Mn in parts per billion per microgram protein was adjusted to 100 (mean ± SE; n = 3 experiments; *p < 0.05 by Student's t test). C, HeLa cells were transfected with the control (Rab5; Control) construct or mock transfected. Two days later, cells were treated with 500 μm Mn for 16 h. Intracellular Mn in parts per billion per microgram protein was then calculated as described in A. Data are from one representative experiment; error bars depict SDs from three ICP-MS runs. Analysis from three independent experiments is presented in D below. D, Quantification of the difference in intracellular Mn between mock-transfected and control-transfected cells from multiple experiments. Cells were transfected and treated with Mn exactly as described in C. In each experiment, the value of the intracellular Mn in parts per billion per microgram protein obtained for mock-transfected cells was adjusted to 100 (mean ± SE; n = 3 experiments; p > 0.05 for the difference between the two groups by Student's t test). E, HeLa cells were transfected with control (Rab5; Control), SLC30A10–WT, or SLC30A10–Δ105-107 constructs. Two days after transfection, the Mn pulse-chase assay was performed as described in Materials and Methods. After the chase, intracellular Mn retained within the cells and released into the medium was measured using ICP-MS. For each transfection condition, the amount of Mn in each compartment (intracellular or extracellular) was expressed as a percentage of the total Mn (i.e., sum of the intracellular and extracellular Mn). As in A, data shown are from one representative experiment; error bars depict SDs from three ICP-MS runs. Analysis from three independent experiments is presented in F. F, Quantification of the difference in intracellular Mn after the pulse-chase assay from multiple experiments. The Mn pulse-chase assay was performed in HeLa cells expressing control (Rab5; Control), SLC30A10–WT, or SLC30A10–Δ105-107 constructs exactly as described in E. In each experiment, for control-transfected cells, the percentage intracellular Mn retained was adjusted to 50 (mean ± SE; n = 3 experiments; *p < 0.05 for the difference between WT and other transfection conditions by one-way ANOVA and Tukey–Kramer post hoc test). G, HeLa cells were transfected with the control Rab5 construct or SLC30A10–WT. Two days after transfection, cells were treated with 100 μm Zn for 16 h. The absolute level of intracellular Zn was then measured using ICP-MS and normalized to total protein. Data shown are from one representative experiment; error bars depict SDs from three ICP-MS runs. Analysis from three independent experiments is presented in H. H, Quantification of the difference in intracellular Zn between cells expressing control (Rab5) or SLC30A10–WT constructs from multiple experiments. Intracellular Zn levels were measured exactly as described in G. In each experiment, for cells expressing the control construct, the absolute value for intracellular Zn (parts per billion per microgram protein) was adjusted to 100 (mean ± SE; n = 3 experiments; p > 0.05 by Student's t test). In this panel, the SE is relatively high because, over multiple experiments, the difference in intracellular Zn levels between control and SLC30A10-transfected cells had a wide distribution. I, Two days after transfection, HeLa cells expressing the control Rab5 construct or SLC30A10–WT were treated with 100 μm Cu for 16 h. After this, intracellular Cu was measured using ICP-MS. Values were normalized to total protein. Data shown are from one representative experiment; error bars depict SDs from three ICP-MS runs. Analysis from three independent experiments is presented in J. J, Quantification of the difference in intracellular Cu between cells expressing control (Rab5) or SLC30A10–WT constructs from multiple experiments. Intracellular Cu was measured as described in I. In each experiment, for cells expressing the control construct, the absolute value for intracellular Cu (parts per billion per microgram protein) was adjusted to 100 (mean ± SE; n = 3 experiments; p > 0.05 by Student's t test).