Fig. 4

TFR1 overexpression aggravated iron overload and mitochondrial dysfunction during AD-iPS cell differentiation. AD-iPS cells were transfected with pcDNA-TFR1 or empty vector, and then were induced to differentiate into neural cells. A, B Western blot assay was performed to measure the expression of TFR1 on day 35 of differentiation. A, C Western blot assay was used to measure the levels of ferritin, FTH1, and FPN on day 35 of differentiation. D Atomic absorption spectrometer was performed to detect intracellular total iron content. E, F Calcein-AM staining was used to detect labile iron pool, and quenching of calcein-AM fluorescence signifies an increase in intracellular labile iron. G ROS level was measured by commercial ROS detection kits. H, I MMP (Δψm) was evaluated by JC-1 staining. J ATP content was accessed using an ATP determination kit. K Mitochondrial staining was used to access mitochondrial fusion and fission. L, M Western blot assay was used to measure the protein levels of DRP1, FIS1, TFR1, MFN2 and OPA1. N = 6. Data from at least three independent experiments were presented as mean ± SEM. **P < 0.01