FIGURE 6.
Cell viability assays to validate the efflux activities of SLC30A10WT and transmembrane domain mutants. A, untransfected HeLa cells were treated with indicated amounts of manganese for 16 h. Cell viability was then assessed as described under “Experimental Procedures.” Viability at 0 mm manganese was set to 100 and used to calculate percent viability at other manganese concentrations (mean ± S.E.; n = 3; *, p < 0.05 for the difference between viability at 0 mm manganese and other manganese concentrations by one-way ANOVA and Dunnett's post hoc test). B, HeLa cells were transfected with a control construct (indicated as Control), SLC30A10WT, or ZnT1WT. Two days after transfection, cells were treated with 0 or 2 mm manganese for 16 h. Viability was then assessed as described under “Experimental Procedures.” For each transfection condition, viability at 0 mm manganese was set to 100 and used to calculate percent viability at 2 mm manganese (mean ± S.E.; n = 4 for each construct; *, p < 0.05 for the difference between control and other groups using one-way ANOVA and Dunnett's post hoc test). C, untransfected HeLa cells were treated with indicated amounts of zinc for 16 h. Further processing was exactly as described in A. Viability at 0 mm zinc was set to 100 and used to calculate percent viability at other concentrations (mean ± S.E.; n = 3; *, p < 0.05 for the difference between 0 mm zinc and other concentrations by one-way ANOVA and Dunnett's post hoc test). D, HeLa cells were processed as described in B, with the only difference being that instead of treatment with 0 or 2 mm manganese the cultures were treated with 0 or 200 μm zinc. For each transfection condition, viability at 0 μm zinc was set to 100 and used to calculate percent viability at 200 μm zinc (mean ± S.E.; n = 3 for each construct; *, p < 0.05 for the difference between control and other groups using one-way ANOVA and Dunnett's post hoc test). E, HeLa cells were transfected with a control construct (denoted as Control), SLC30A10WT, or various transmembrane domain mutants. After 2 days, cells were treated with 0 or 2 mm manganese for 16 h. Cell viability was then assessed. For each transfection condition, viability at 0 mm manganese was set to 100 and used to calculate percent viability at 2 mm manganese (mean ± S.E.; n ≥ 4 per construct; *, p < 0.05 for the difference between WT and other transfection conditions by one-way ANOVA and Dunnett's post hoc test). One-way ANOVA and Dunnett's post hoc test also revealed that there was no difference between control and D248A, E25A, N127A,H244A, or D40A groups (p > 0.05); however, there was a significant difference between control and WT, N43A, or D47A groups (p < 0.05).