Fig. 6. Impaired balance of mitochondria fission and fusion along with defective autophagy in the PS1-E120K iPSC-derived neurons. (A) Representative western blot image from three independent experiments. (B~I) Quantification of marker proteins associated with mitochondrial fission (Drp1, p-Drp1 and Fis1) and fusion (OPA1, Mfn1and Mfn2). Note that fission-related proteins were increased, whereas fusion-related proteins were decreased in the PS1-E120K cells compared with the control cells. Mitophagy-related protein PINK1 and PARKIN showed dramatic increases in the PS1-E120K cells compared with the control cells. (J~K) Q-PCR analysis revealing the mRNA expression of fission and fusion-related genes in 10 weeks differentiated neurons from iPSCs. (L) Representative western blot image from three independent experiments. (M~Q) Quantification of the expression of ubiquitination (Ub) and autophagy-related proteins (p62, Beclin1, LC3B, LAMP2). Note that levels of Ub and LC3B were significantly increased, whereas the LAMP2 signal was significantly decreased in the PS1-E120K iPSC-derived neurons. (R) Immunocytochemical staining showing the expression of LC3b (red) and DAPI (blue) in both iPSC-derived neurons at 10 weeks of neuronal differentiation. Note that PS1-E120K iPSC-derived neurons exhibit a dramatic increase of LC3b in the periphery of soma area (arrow), compared with the control group. (S) Autophagy flux assay showing a significant increase in LC3B-II expression after treatment with 10 uM chloroquine (CQ) in the PS1-E120K iPSC-derived NPC compared with the control. (T~P) CytoID (green) stained with DAPI (blue) after CQ treatment showing a dramatic increase in number of autophagic vesicles in the PS1-E120K iPSC-derived NPC compared with the control. Representative western blot and ICC images were obtained from three independent experiments. Scale bar: 10 µm.
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