Figure 2.

Overexpression of SLC30A10-WT or SLC30A10-T95I in HeLa cells reduces Mn levels and protects against Mn toxicity. A: HeLa cells were either left untransfected (−) or transiently transfected with FLAG-SLC30A10-WT construct (+) and harvested 48 h after transfection. Immunoblots were performed to detect SLC30A10 (using the custom anti-SLC30A10 antibody), FLAG or tubulin. B: immunoblots were performed to detect SLC30A10 (using the anti-SLC30A10 antibody) or tubulin from HeLa clones expressing SLC30A10-WT or SLC30A10-T95I. Relative expression of SLC30A10-WT or T95I, normalized to tubulin, respectively is 1.00 ± 0.008 and 0.655 ± 0.096 (means ± SE; n = 3; P < 0.05 by t test). C–F: mock-infected HeLa cells (control) or clonal HeLa cells expressing SLC30A10-WT or SLC30A10-T95I were treated with 250 µM Mn for 16 h. Intracellular levels of Mn (C), Fe (D), Cu (E), or Zn (F) were measured by inductively coupled plasma-mass spectrometry (ICP-MS) and normalized to total cell counts (means ± SE; n = 4–5; *P < 0.05 and n.s. denotes not significant for indicated comparisons by one-way ANOVA and Tukey’s post hoc test). G: viability of cells infected as described in C–F was assessed 16 h after treatment with indicated Mn doses. For each infection condition, viability at 0 mM Mn was independently set to 100 (means ± SE; n = 4, *P < 0.05 by two-way ANOVA and Tukey’s post hoc test with a and b indicating differences in comparison with control or SLC30A10-WT infection conditions, respectively, at each Mn concentration). WT, wild type.