Fig. 2. Homozygous R136S mutation protects against APOE4-induced p-Tau accumulation in human neurons.
a–d, Representative western blot images (a) and quantification of APOE (b), PHF1+ p-Tau (c) and AT8+ p-Tau (d) levels in lysates of E4, E3, E4-S/S or E4-R/S neurons. In b, APOE levels were normalized to those of E4. TUJ1 was used as loading control (E4, n = 32; E3, n = 31; E4-S/S, n = 64 (n = 24 from E4-S/S-A; n = 8 from E4-S/S-B; n = 32 from E4-S/S-C); E4-R/S, n = 59 (n = 31 from E4-R/S-A; n = 28 from E4-R/S-B)). In c, PHF1+ p-Tau levels were normalized to those of E4. TUJ1 was used as loading control (E4, n = 32; E3, n = 31; E4-S/S, n = 64 (n = 24 from E4-S/S-A; n = 8 from E4-S/S-B; n = 32 from E4-S/S-C); E4-R/S, n = 59 (n = 31 from E4-R/S-A; n = 28 from E4-R/S-B)). In d, AT8+ p-Tau levels were normalized to those of E4. TUJ1 was used as loading control (E4, n = 28; E3, n = 27; E4-S/S, n = 55 (n = 16 from E4-S/S-A; n = 11 from E4-S/S-B; n = 28 from E4-S/S-C); E4-R/S, n = 59 (n = 31 from E4-R/S-A; n = 28 from E4-R/S-B)). e, Representative images showing immunostaining of p-Tau (PHF1), total Tau and MAP2 in E4, E3, E4-S/S or E4-R/S human neurons, with some PHF1+ puncta (white arrowheads in insets). f,g, Quantification of fraction of the PHF1+ area over MAP2+ area (f) and fraction of PHF1+ puncta area over MAP2+ area (g) (E4, n = 25 fields of view; E3, n = 25 fields of view; E4-S/S, n = 38 fields of view; E4-R/S, n = 33 fields of view). The ratio of PHF1+ area (f) and PHF1+ puncta area (g) over MAP2 area was normalized to that of E4. Western blot data (b–d) were made up of at least three independent rounds of differentiation, and all data were combined. Scale bars (e), 20 µm. In b–d, n = biological replicates. Throughout, data are expressed as mean ± s.e.m. Differences between groups were determined by Welch’s ANOVA followed by Dunnett’s T3 multiple comparison test (b–d,g) or ordinary one-way ANOVA followed by Tukey’s multiple comparison test (f). Comparisons of P ≤ 0.05 are labeled on the graph.