Figure 5

Mn²⁺ induces ferritin degradation and Fe²⁺ chelation exacerbates Mn²⁺-mediated JNK activation.

(a) Representative western analysis of ferritin levels upon Fe²⁺ and Mn²⁺ treatment. 0.5 and 1 mM of Fe²⁺ for 24 h resulted in a concentration-dependent increase in ferritin levels in S-DMT1 cells. In contrast, both 0.5 and 1 mM Mn²⁺ reduced ferritin levels compared to untreated. Ferritin loss associated with 0.5 mM Mn²⁺ was partially reversed when co-treated with 0.5 mM Fe²⁺. (b) Representative western analysis of S-DMT1 cells pretreated with 0.5 mM Fe²⁺ for 12 h (to upregulate ferritin) before treatment with 0.5 mM Mn²⁺ for another 12 h, with or without autophagy inhibition. In Fe²⁺ pretreated cells, incubation with Mn²⁺ significantly (**p < 0.01) reduced ferritin levels compared to Fe²⁺ treatment alone. With NH₄Cl, Mn²⁺-induced reduction of ferritin is significantly suppressed (*p < 0.05) compared to Mn²⁺ without inhibitor. Corresponding bar chart shows ferritin protein fold change over Fe²⁺ treatment (n = 3, Student’s t-test), with error bars representing standard error of mean (S.E.M.). (c) Increasing concentration of Fe²⁺ led to marked ferritin accumulation in autophagy deficient ATG5^−/− MEF compared to ATG5^+/+. Autophagy deficiency in the ATG5^−/− MEF was verified through the increase in mitochondrial SOD2 compared to ATG5^+/+ MEF. (d) Mn²⁺ treatment together with Fe²⁺ chelation by 1,10-phenanthroline for 12 h resulted in increased JNK phosphorylation compared to Mn²⁺ alone (**p < 0.01, n = 4, Student’s t-test). In contrast, phenanthroline in the presence of Fe²⁺ did not result in increased JNK phosphorylation due to sufficient Fe²⁺. Similarly, addition of Fe²⁺ to Mn²⁺/phenanthroline reduced JNK phosphorylation (*p < 0.05).