Figure 6.

After clonal selection of SLC30A10-WT or SLC30A10-T95I expression in ΔSLC30A10 HepG2 cells, rescue activity of the SLC30A10-T95I mutant is comparable to SLC30A10-WT. A: immunoblot analyses were performed to detect SLC30A10 (using the custom anti-SLC30A10 antibody) or tubulin in ΔSLC30A10 HepG2 cells infected a second time with SLC30A10-WT or SLC30A10-T95I. Cells were clonally selected after the second infection. Relative expression of SLC30A10-WT or T95I, normalized to tubulin, respectively is 1.00 ± 0.239 and 0.835 ± 0.085 (means ± SE; n = 3; P > 0.05 by t test). B–E: intracellular metal measurements were performed in mock-infected HepG2 cells (control) or ΔSLC30A10 HepG2 cells that underwent a second mock, SLC30A10-WT or SLC30A10-T95I infection (ΔSLC30A10 + control; ΔSLC30A10+SLC30A10-WT; or ΔSLC30A10+SLC30A10-T95I, respectively). ΔSLC30A10 cells infected with SLC30A10-WT or SLC30A10-T95I underwent clonal selection. Cells were treated with 125 µM Mn for 16 h before analyses. Metal levels were normalized to total cell counts (means ± SE; n = 5–7, *P < 0.05 and n.s. denotes not significant by one-way ANOVA and Tukey’s post hoc test). F: viability of cells infected and clonally selected as described in B–E was assayed after 16 h treatment with indicated Mn doses. For each cell line, viability at 0 mM Mn was set to 100 (means ± SE; n = 5, *P < 0.05 using two-way ANOVA and Tukey’s post hoc test with a and b indicating differences in comparison with control or ΔSLC30A10 + control groups, respectively at each Mn dose. There were no differences between ΔSLC30A10+SLC30A10-WT or ΔSLC30A10+SLC30A10-T95I groups at any Mn concentration by two-way ANOVA). WT, wild type.