Figure 2

Uptake and cytoplasmic accumulation of Mn²⁺.

Calcein quenching assay was performed on S-DMT1 cells treated with either Mn²⁺ or Fe²⁺ for 30 min. The effect of cytoplasmic accumulation on cell viability was determined using MTT assay. (a) Mn²⁺ treatment reduced calcein fluorescence in a concentration-dependent manner (compared to untreated, **p < 0.01; ***p < 0.001, n = 4, one-way ANOVA, Dunnett post hoc test). (b) Fe²⁺ treatment reduced calcein fluorescence (compared to untreated, *p < 0.05, **p < 0.01, ***p < 0.001, n = 4) but not in a concentration-dependent manner. (c) MTT assay of Mn²⁺ treatment of S-DMT1 cells for 24 h reduced cell viability compared to vector cells, especially for 0.5 and 1 mM Mn²⁺ (*p < 0.05, **p < 0.01, n = 4, two-way ANOVA, Bonferroni post hoc test). (d) MTT assay of Fe²⁺ treatment of S-DMT1 cells for 24 h did not show any significant reduction in cell viability compared to vector cells (n = 4). (e) Representative western blot showing upregulation of ferritin heavy chain (FTH1) protein in a Fe²⁺ concentration-dependent fashion in S-DMT1 cells treated for 12 h. Corresponding bar chart displays ferritin protein levels with Fe²⁺ treatment (**p < 0.01, ***p < 0.001, n = 3), with error bars representing standard error of mean (S.E.M.). β-actin was used as a loading control.