Fig. 3. Increase of Aβ and p-tau levels in the APP-V715M iPSC-derived neurons. (A) ELISA detection of the extracellular Aβ₄₂ and Aβ₄₀ levels secreted from the iPSC-derived neurons into the medium. (B) Immunocytochemical analysis showing the expression of Aβ deposits using an antibody against Aβ₄₂ (red), co-stained with Tuj1 (green) and DAPI (blue) at 10 weeks of neuronal differentiation. The fourth figures (from the left) show the z-stack images of the Aβ₄₂-positive Aβ deposits (indicated as arrows) in the APP-V715M iPSC-derived neurons. (C) The TBS-insoluble/SDS-soluble intracellular Aβ₄₂ and Aβ₄₀ levels were measured in a total 1 µg of proteins using ELISA at 10 week-differentiated neurons. (D~F) Quantification of the expression level of total APP and Aβ oligomers. Note that levels of Aβ oligomers were increased in the APP-V715M iPSC-derived neurons. (G) Immunocytochemical analysis showing the expression of AT8 (p-tau) (red) and MAP2 (green), counter-stained with DAPI (blue) in the iPSC-derived neurons at 10 weeks of neuronal differentiation. Expression of AT8 (p-tau) was indicated in the soma (arrow) and the neurites (arrowhead) (H~I) Quantification of the immunocytochemical analysis, normalized against MAP2-positive cells. (J~L) Western blot analysis showing an increase of AT8 and the ratio of AT8/Tau5 in the APP-V715M iPSC-derived neurons. Scale bar: 10 µm. Data are presented as mean±SEM. Sample sizes: Fig. 3A, C, E, F, K and L (n=3); Fig. 3H and I (n=7). Two-tailed Student's t test was used for statistical analysis. ^*p<0.05; ^**p<0.01; and ^***p<0.001.