Confirmation of cell transfection. (A) A representative western blot is shown demonstrating FLAG-tagged HFE expression in transfected SH-SY5Y cells (B) Intracellular labile iron was determined in the differentiated SH-SY5Y cells using the cell-permeable dye calcein-AM, as described in Methods. Fluorescence of calcein is quenched in the presence of iron, thus fluorescence is inversely proportional to labile iron levels. Intracellular labile iron levels were in the order C282Y > H63D = Vector > Wt. Results are reported here as the average ± SEM of three independent assays. * and *** indicate p < 0.05 and p < 0.001, respectively, compared to Wt.