Amot depletion in cultured hippocampal neurons affects dendritic tree organization.

(A, B) Validation of Amot-knockdown efficiency in neurons by qRT-PCR (n = 3/group; p = 0.0041) (A) and western blot (B). (C) Rat hippocampal neurons were transfected on DIV7 with an empty pLKO plasmid (control), a plasmid with Amot shRNA, and a plasmid with Amot shRNA and a plasmid that expressed mouse Amot–GFP (Amot shRNA + Amot; rescue experiment). (D, E) Quantification of TDL (D) and Sholl analysis (E) of hippocampal neurons that were transfected with plasmids as in (C) or transfected with Amot–GFP plasmid. Control: n = 60; Amot: n = 38; Amot shRNA: n = 60; Amot shRNA + Amot: n = 58. To Control; p = 0.2678, p p Amot fl/fl mice that were transfected with a Cre-expressing plasmid. (G, H) Quantification of TDL (G) and Sholl analysis (H) of dendritic trees of neurons from (F). Control vector: n = 33; Cre plasmid: n = 40. p Amot fl/fl hippocampal neurons led to the down-regulation of Amot expression, determined by western blot. (J) Domain architecture of Amot protein. The Amot ID domain contains LPXY and PPXY motifs (red) that bind Yap1 and the AB (blue). (K) Quantification of TDL of Amot-knockdown rat hippocampal neurons that were transfected with the indicated Amot constructs. Control cells were transfected with an empty pLKO vector. Control: n = 107; Amot shRNA: n = 80; Amot shRNA + Amot: n = 100; Amot shRNA + Amot ID domain: n = 37; Amot shRNA + Amot ΔID domain: n = 45; Amot shRNA + Amot ΔAB: n = 59. To control; p p p = 0.0064, p = 0.0015, p p = 0.0047, p = 0.0033, p = 0.09695. Images were obtained from at least three independent cultures. Statistical significance was analyzed using two-tailed and unpaired t tests (A, G), one-way analysis of variance followed by Tukey’s post hoc test (D, K), and two-way analysis of variance followed by Bonferroni’s test (E, H). Numerical values that underlie the graphs are shown in S1 Data. See S1 Table for detailed statistics for E and H. **p p < 0.0001. Bars represent the mean ± SEM. Scale bars = 50 μm. AB, actin-binding motif; Amot, angiomotin; CC, coiled-coil domain; DIV, day in vitro; GFP, green fluorescent protein; ID, intrinsic disordered; ns, not significant; PR, proline-rich region; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean; shRNA, short-hairpin RNA; TDL, total dendrite length; Yap1, Yes-associated protein 1.