Fig. 5

Knockdown of IGFBP3 decreases Aβ peptides in APOE ε4-expressing AD patient and APOE ε3/4 patient iNs at amyloid-seeding stage. a Validation of IGFBP3 expression between PSEN1-harboring AD iNs expressing APOE ε4 from the amyloid-seeding stage and from the amyloid oligomer-progressive stage. Data represent mean ± SEM. ANOVA-test, **P < 0.01; n = 4 per sample. b Quantitative RT-PCR analysis of IGFBP3 expression in AD patient iNs derived from APOE ε3/4 patient fibroblasts (AG05810 and AG04402), PSEN1 patient fibroblasts (AG06848), PSEN2 patient fibroblasts (AG09908), and sporadic AD patient fibroblasts (AG06869). Data represent mean ± SEM. ANOVA-test, **P < 0.01; n = 4 per sample. c, d Western blot analysis of IGFBP3 in AD-patient-derived iNs treated with IGFBP3-shRNA. Data represent mean ± SEM. ANOVA-test, **P < 0.01; n = 3 per sample. e Immunofluorescence of EEA1 and  Aβ (6E10) in AD patient (PSEN1 mutation) iNs treated with IGFBP3 knockdown. APOE ε4 expression was induced by doxycycline at amyloid-seeding or amyloid-progressive stage. Scale bars, 10 µm. f, g Immunofluorescence of EEA1 and  Aβ (6E10) in familial AD patient (f, APOE ε3/3 genotype) or sporadic AD patient (g, APOE ε3/3 genotype) cell line treated with IGFBP3 knockdown. Scale bars, 10 µm. h Quantification of EEA1- and  Aβ-positive puncta in AD patient iNs harboring PSEN1 mutation treated with IGFBP3-shRNA. Data represent mean ± SEM. ANOVA-test, **P < 0.01; n = 5 per sample. i Quantification of EEA1- and  Aβ-positive puncta in familial AD patient harboring PSEN2 mutation (left, APOE ε3/3 genotype) or sporadic AD patient (right, APOE ε3/3 genotype) cell line treated with IGFBP3 knockdown. Expression of APOE ε4 was induced by doxycycline at amyloid-seeding or amyloid-progressive stage. Data represent mean ± SEM. ANOVA-test, *P < 0.05; n = 5 per sample. j Western blot analysis shows the decrease of Aβ oligomers in APOE ε4-expressing AD patient iNs harboring PSEN1 mutation treated with IGFBP3-shRNA. Data represent mean ± SEM. ANOVA-test, *P < 0.05; n = 4 per sample. k Immunostaining of EEA1 and  Aβ in AD patient iNs harboring APOE ε3/4. Knockdown or overexpression of IGFBP3 was treated in the culture before the amyloid initial phase. Scale bar, 20 µm. l, m Quantification of EEA1- and  Aβ-positive puncta in AD patient iNs harboring APOE ε3/4 mutation. Data represent mean ± SEM. ANOVA-test, *P < 0.05, **P < 0.01; n = 5 per sample. + APOE4 (day 7): AD patient iNs expressing APOE ε4 from day 7; + APOE4 (day 14): AD patient iNs expressing APOE ε4 from day 14; -APOE4: AD patient iNs with no APOE ε4 expression