FIGURE 4.

Inhibition of γ-secretase activity reduced ferritin levels in response to iron treatment in wild-type fibroblasts. Wild-type fibroblasts were treated with or without 5 μM DAPT in DMEM containing 10% FBS after seeding. After 1 h, ferrous sulfate was added and the cells were incubated at 37°C in 5% CO₂ for 72 h. (A) Western blot analysis of ferritin light chain in wild-type fibroblasts treated with or without ferrous sulfate or DAPT. (B) Quantification of intracellular ferritin light chain levels. Ferritin light chain levels at 10 μM ferrous sulfate treatment (without DAPT) were normalized to α-tubulin protein levels and adjusted to 100%. The relative change of ferritin light chain at each concentration was calculated. DAPT treatment significantly inhibited intracellular ferritin light chain levels. n = 3, from 3 independent experiments, ^**P< 0.01, ^***P< 0.001, one-way ANOVA followed by Tukey’s multiple-comparison tests. (C) Western blot analysis of ferritin heavy chain in wild-type fibroblasts treated with or without ferrous sulfate or DAPT. (D) Quantification of intracellular ferritin heavy chain levels. Ferritin heavy chain levels in wild-type fibroblasts with 10 μM ferrous sulfate treatment (without DAPT) were normalized to α-tubulin protein levels and adjusted to 100%. The relative change of ferritin light chain at each concentration was calculated. DAPT treatment significantly inhibited intracellular ferritin heavy chain levels. n = 3, from 3 independent experiments, ^***P< 0.001, ^****P< 0.0001, one-way ANOVA followed by Tukey’s multiple-comparison tests. ■, ferrous sulfate treatment. 🌑, DAPT and ferrous sulfate treatment. All quantitative data shown as mean±SEM. Ferritin-L, ferritin light chain. Ferritin-H, ferritin heavy chain.