Figure 6.

Extracellular oxidized Cp favors intracellular iron accumulation. A, SH-SY5Y neuroblastoma cells maintained in medium supplemented with 10% FBS (CTR) were iron loaded by treatment (20 h) with FAC (100 μm) and Asc (200 μm). Extracellular iron and ascorbate were subsequently removed (w/o FAC+Asc) and cells further incubated (24 h) in the medium alone without serum (No Cp) or in the presence either of functional Cp (5 μg/ml) or of inactive oxidized Cp (Ox-Cp). Cell lysates resolved by SDS-PAGE were used for WB performed with anti-Ferritin (Frt) and anti-Ferroportin (Fpn); reactivity of anti-β-tubulin (Tub) was used for signal normalization. B, Analysis of Ferritin OD normalized signals (in arbitrary units) in treated SH-SY5Y. C, Evaluation of Fe retained in the cells. SH-SY5Y cells were iron loaded as previously described but with 95 μm FAC supplemented with ⁵⁵Fe-citrate (5 μm) as a tracer. After 20 h incubation, the cells were washed, and one aliquot (2 × 10⁵ cells/well, in triplicate) was kept for 100% iron-loading evaluation (FAC), while other aliquots were further incubated (24 h) in serum-free medium alone (No Cp), or in the presence either of functional Cp or of inactive oxidized Cp (Ox-Cp). Cells were then washed, lysed, and counted. Radioactive counts per minute in each well (in triplicate for each condition) were normalized on the basis of total protein contents and used to evaluate the differences in percentages of total intracellular iron contents. D, Evaluation of Fe released by the cells. Spent media after 24 h incubation in serum-free medium alone (No Cp), or in the presence either of functional Cp or of inactive oxidized Cp (Ox-Cp), were collected and counted. Radioactive counts per minute of medium from each well (in triplicate for each condition) were used for the calculation of percentages of iron released from total intracellular iron loading. Data in B–D were analyzed by both unpaired ANOVA and Student's t test; respective means with SE of five (B) and three (C, D) independent experiments are indicated (p values: *p < 0.05; **p < 0.005; ***p < 0.0001).