Fig. 6

GSK3B reversed the protective effects of TFR1 knockdown on iron overload and mitochondrial dysfunction during AD-iPS cell differentiation. AD-iPS cells were transfected with sh-TFR1 alone or together with pcDNA-GSK3B, and then were induced to differentiate into neural cells. expression, A, B Western blot assay was performed to examine the protein level of GSK3B on day 35 of differentiation. A, C Western blot assay was used to evaluate the levels of ferritin, FTH1, and FPN. D Atomic absorption spectrometer was used to evaluate intracellular total iron content. E, F Calcein-AM staining was performed to measure labile iron pool, and quenching of calcein-AM fluorescence signifies an increase in intracellular labile iron. G ROS level was examined with commercial ROS detection kits. H, I MMP (Δψm) was evaluated by JC-1 staining. J ATP content was determined 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