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. 2017 Oct 17;114(45):E9645–E9654. doi: 10.1073/pnas.1707151114

Fig. 5.

Fig. 5.

Cytoplasmic tau is important for mislocalization and insolubilization of BRCA1. (A) SH-SY5Y cells transfected with wild type (WT) or P301L mutant 4-repeat tau were treated with in vitro-aggregated tau seeds [either full length (FL) or C-terminal fragment (251)]. After fractionation, supernatant (sup) or pellet (ppt) was subjected to Western blot. (B) Quantification of BRCA1 in the pellet fraction. Mean ± SEM: 1.00 ± 0.09 in WT, 1.08 ± 0.08 in P301L, 0.87 ± 0.11 in WT + FL, 0.95 ± 0.09 in WT + 251, and 1.74 ± 0.15 in P301L + FL. (C) Immunocytochemical images of seed-dependent tau aggregation colabeled with DAPI, pTau, and BRCA1 antibodies. (D) Representative images of Aβ staining of 3×Tg-AD mice hippocampal region. (E–G) Representative immunohistochemical images of 3×Tg-AD mice hippocampus by anti-pTau (E), anti-γ-H2ax (F), and BRCA1 (G) staining. Samples are from 3×Tg-AD mice at 3, 6, 9, and 12 mo of age. n = 10–15 visual fields from six to seven mice for 3×Tg-AD mice. Quantitation of positive cells for each protein are shown at the Right side of the images. Statistical significance was determined by the Tukey method. Mean ± SEM: pTau: 33.26 ± 2.57/mm in 3 mo, 37.30 ± 1.99/mm in 6 mo, 44.56 ± 3.19/mm in 9 mo, and 55.12 ± 2.69/mm in 12 mo; γ-H2ax: 1.28 ± 0.22/mm in 3 mo, 3.23 ± 0.90/mm in 6 mo, 4.14 ± 0.66/mm in 9 mo, and 7.97 ± 0.90/mm in 12 mo; BRCA1: 0.56 ± 0.21/mm in 3 mo, 1.07 ± 0.24/mm in 6 mo, 3.41 ± 0.33/mm in 9 mo, and 6.94 ± 0.77/mm in 12 mo. (H and I) Expression of BRCA1 in detergent fractionated the cortical (H) and hippocampal (I) regions of 3×Tg-AD mice at 12 mo of age. n = 2 mice at 12 mo of age. Representative blot was shown. (J) Microscopic images of comet assays. Representative images are shown from a total of n = 136 cells from WT and 3×Tg-AD animals. Insets are magnified nuclei. (K) Quantification of tail DNA% in E. Solid circles are WT, and open circles, 3×Tg-AD mice. Gray bars represent means. Statistical significance was determined using the Holm–Sidak method. Mean ± SEM: WT: 36.71 ± 1.03% in 3 mo, 32.11 ± 0.92% in 6 mo, 36.55 ± 0.92% in 9 mo, and 36.32 ± 0.98% in 12 mo; 3×Tg-AD: 34.10 ± 0.88% in 3 mo, 33.67 ± 0.92% in 6 mo, 41.24 ± 0.90% in 9 mo, and 40.87 ± 1.65% in 12 mo. *P < 0.05.