Fig. 3

TFR1 knockdown attenuated iron overload and mitochondrial dysfunction during AD-iPS cell differentiation. AD-iPS cells were transfected with sh-TFR1 or sh-NC, and then were induced to differentiate into neural cells. A, B Western blot assay was performed to access TFR1 level on day 35 of differentiation. A, C Western blot assay was performed to access ferritin, FTH1, and FPN levels on day 35 of differentiation. D Intracellular total iron was evaluated using atomic absorption spectrometer. E, F Calcein-AM staining was conducted to detect the labile iron pool, and quenching of calcein-AM fluorescence signifies an increase in intracellular labile iron. G ROS level was detected using commercial ROS detection kits. H, I MMP (Δψm) was evaluated by JC-1 staining. J ATP content was measured 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 were accessed with Western blot assay. N = 6. Data from at least three independent experiments were presented as mean ± SEM. **P < 0.01